Methods for improving immunological response in vaccinated animals

ABSTRACT

A method is provided for increasing an immunological response to a target antigen in an animal by administering an immunogenic amount of a vaccine comprising a polypeptide conjugate comprising the target antigen conjugated to a carrier polypeptide by means of a linker polypeptide which is rich in predicted linear B-cell epitopes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/831,602, filed Aug. 20, 2015, issued as U.S. Pat. No. 10,441,652 onOct. 15, 2019, which claims priority to U.S. Provisional Application No.62/039,977, filed Aug. 21, 2014, entitled “Methods for ImprovingImmunological Response in Vaccinated Animals”, each of which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

A method is provided for increasing an immunological response to atarget antigen in an animal by administering an immunogenic amount of avaccine comprising a polypeptide conjugate comprising the target antigenconjugated to a carrier polypeptide by means of a linker polypeptidewhich is rich in predicted linear B-cell epitopes.

BACKGROUND OF THE INVENTION

Conjugated vaccines rely on antigen attachment to a carrier protein orvector, allowing an otherwise non-immunogenic antigen to take on theimmunogenicity of the carrier. Conjugated vaccines employing thewidely-used tetanus toxoid carrier have been shown to have reducedefficacy in individuals previously exposed to tetanus toxoid, aspre-existing immunity against the carrier neutralizes the conjugatedvaccine. Schutze et al., Carrier-induced epitopic suppression, a majorissue for future synthetic vaccines. J. Immunol. 1985; 135(4):2319-22.Other common carriers, such as adenovirus, are also likely to beneutralized by pre-existing immunity, especially upon repeatedvaccinations or booster injections. Pinto et al., Induction of CD8+ Tcells to an HIV-1 antigen through a prime boost regimen withheterologous E1-deleted adenoviral vaccine carriers. J. Immunol. 2003p;171(12):6774-9.

Methods are provided herein for improving a specific immune response ina subject to a target antigen having low predicted linear B-cell epitopescoring, the method comprising administering to the subject apolypeptide conjugate comprising the target antigen covalently attachedto a carrier polypeptide by means of an intervening linker polypeptide,wherein the linker polypeptide and/or the carrier polypeptide exhibithigher predicted linear B-cell epitope scoring than the target antigen,and wherein the carrier polypeptide does not stimulate a substantialT-cell response.

SUMMARY OF THE INVENTION

Embodiments of the present invention provide novel methods for improvingimmunological response to a target antigen in a polypeptide conjugate orfusion protein.

In some embodiments, a method is provided for stimulating an improvedimmunological response to a target antigen in an animal, the methodcomprising administering an immunogenic amount of a vaccine to ananimal, the vaccine comprising a polypeptide conjugate comprising (a) acarrier polypeptide comprising an inactivated chloramphenicol acetyltransferase (CAT) enzyme; (b) a linker polypeptide, and (c) a targetantigen, wherein the carrier polypeptide does not stimulate asubstantial T cell response. In some embodiments, the target antigenexhibits low % predicted linear B-cell epitopes.

In some embodiments, the target antigen exhibits low % predicted linearB-cell epitopes selected from <10%, <7%, <5%, <4%, <3%, <2%, <1%, <0.1%or 0% predicted linear B-cell epitopes, wherein the % predicted linearB-cell epitopes for the target antigen is determined by

(1) generating a BepiPred 1.0 predicted Linear B-cell epitope score foreach amino acid in the amino acid sequence of the target antigen;

(2) counting the amino acids for which the BepiPred 1.0 predicted LinearB-cell epitope score is above a threshold value;

(3) dividing the number of amino acids above the threshold value by thenumber of amino acids in the target antigen to get a fraction; and

(4) multiplying the fraction by 100 to obtain the % predicted linearB-cell epitopes for the target antigen.

In some embodiments, the linker polypeptide exhibits high % predictedlinear B-cell epitopes. In some embodiments, the linker polypeptideexhibits high % predicted linear B-cell epitopes selectedfrom >50%, >60%, >70% or >80% predicted linear B-cell epitopes, whereinthe % predicted linear B-cell epitopes in the linker polypeptide isdetermined by

-   -   (1) generating a BepiPred 1.0 predicted Linear B-cell epitope        score for each amino acid in the amino acid sequence of the        linker polypeptide;    -   (2) counting the amino acids in the linker polypeptide for which        the BepiPred 1.0 predicted Linear B-cell epitope score is above        a threshold value;    -   (3) dividing the number of amino acids above the threshold value        by the total number of amino acids in the linker polypeptide to        get a fraction; and    -   (4) multiplying the fraction by 100 to get the % predicted        Linear B-cell Epitopes for the linker polypeptide.

In some embodiments, a polypeptide conjugate is provided comprising acarrier polypeptide comprising an amino acid sequence selected from SEQID NO: 3, 7, 8, 26, 27, 28, or 29.

In some embodiments, a polypeptide conjugate is provided comprising alinker polypeptide that comprises an amino acid sequence selected fromSEQ ID NO: 10, 11, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 53, 54, 55,56 or 57.

In some embodiments, methods are provided for stimulating an improvedimmunological response to a target antigen in an animal, wherein theimproved immunological response comprises one or more of (1) astatistically significant increase in serum anti-target antigen IgG orserum anti-carrier IgG from the animal observed after the first orsubsequent administration of the vaccine comprising the polypeptideconjugate, compared to control immunized with target antigen and/orcarrier without the linker polypeptide, (2) a statistically significantincrease or decrease in a specific effect in the animal followingadministration of the vaccine comprising the polypeptide conjugatecompared to a control, or (3) a statistically significant fasterstatistically significant immunological response or specific effect inthe animal following administration of the vaccine comprising thepolypeptide conjugate compared to a control.

In some embodiments, the improved immunological response in the animalcomprises a statistically significant faster immunological response orspecific effect compared to a control, wherein the response or effectoccurs within six days, five days, four days, three days, two days, orwithin one day following the first or subsequent administrations to theanimal of the vaccine comprising the polypeptide conjugate.

In some embodiments, the specific effect is selected from increased milkproduction, increased body weight, increased lean body weight, decreasedpercent body-fat, or decreased body weight in the animal compared to acontrol without immunization.

In some embodiments, the immunogenicity of said target antigen in thepolypeptide conjugate is increased as compared to the target antigen inthe absence of the polypeptide conjugate.

In some embodiments, the target antigen is selected from somatostatin14, gonadotropin releasing hormone (GnRH), luteinizing hormone releasingfactor, calcitonin neuropeptide, myostatin, HIV envelope protein,tuberculosis outer membrane protein, Neisseria meningitides outermembrane protein complex; or a fragment thereof.

In some embodiments, the target antigen fragment is a polypeptide from 5to 40 amino acids in length.

In some embodiments, the target antigen comprises an amino acid sequenceconsisting of one or more of SEQ ID NOs: 1, 32, 33, 34, 35, 36, 37, 38,40, 41, 43, 44, 46, 47, 48, 49, 50, 51, or 52.

In some embodiments, the target antigen comprises an amino acid sequenceconsisting of SEQ ID NO: 1.

In some embodiments, the target antigen is not somatostatin 14.

In some embodiments, a method is provided for rapidly increasing milkproduction in an animal, the method comprising: (1) administering animmunogenic amount of a vaccine to an animal, the vaccine comprising apolypeptide conjugate comprising a target antigen that issomatostatin-14 attached to an inactivated chloramphenicol acetyltransferase (CAT) enzyme by a linker polypeptide, wherein milkproduction is significantly increased relative to that of a controlanimal that does not receive administration of said vaccine. In someaspects, the animal is a dairy cow, beef cow, goat, or sow. In someaspects, the milk production is increased within 4 days of theadministration of the vaccine. In some aspects, peak milk production isachieved within 8-14 days of the administration of the vaccine. In someaspects, the increase in milk production persists for at least 21 days.

In some embodiments, a method is provided for rapidly increasing leanmeat production in an animal, the method comprising: (1) administeringan immunogenic amount of a vaccine to the animal, the vaccine comprising(a) a polypeptide conjugate comprising somatostatin-14 covalentlyattached to an inactivated CAT enzyme by a linker polypeptide, and (2)subsequently administering at least one additional immunogenic amount ofthe vaccine, wherein subsequent administration does not produce ananamnestic response to the carrier, and wherein lean meat production isincreased relative to that of an animal that does not receive saidadministration.

In some aspects, wherein the vaccine is administered at least 3 times.In some aspects, the vaccine is administered at intervals of 21 days. Insome aspects, the animal is a pig or cow.

In some embodiments, a method is provided for developing a conjugatedvaccine, comprising selecting a target antigen exhibiting low %predicted B-cell epitopes; and covalently attaching the target antigento a carrier polypeptide via a linker polypeptide exhibiting high %predicted linear B-cell epitopes to form a polypeptide conjugate;wherein the carrier polypeptide does not stimulate a substantial T cellresponse. In some aspects, the target antigen exhibits lower % predictedB-cell epitopes than the linker polypeptide. In some aspects, theBepiPred 1.0 predicted Linear B-cell epitope score threshold is 0.2. Insome aspects, the linker polypeptide is heterologous to the targetantigen.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing BepiPred 1.0 predicted linear B epitope scoresof four different linker sequences, in accordance with embodimentsdescribed herein.

FIG. 2 is a graph showing that the B epitope predicted scores of alinker described herein having four repeated Pro-Arg sequences arehigher than for a single Pro-Arg sequence.

FIG. 3 is a graph showing the milk increase of vaccinated cows by eachvaccination as compared to control cows in accordance with aspects ofthe present invention. A rapid increase in average milk production isexhibited following administration of the vaccine comprising thepolypeptide conjugate having amino acid sequence of SEQ ID NO: 13.

FIG. 4 is a graph showing the mean actual difference in milk productionbetween vaccinated and control cows, in accordance with aspects of thepresent invention.

FIG. 5A is a graph showing the increase in pig body weight over a 12week study in accordance with aspects of the present invention.

FIG. 5B provides an improved view of the final four weeks in accordancewith aspects of the present invention.

FIG. 5C shows the difference in mean body weight between vaccinated andcontrol pigs, in accordance with aspects of the present invention.

FIG. 6 is a graph showing the comparative serological IgG responses toSST-CAT of the rCAT experimental vaccinated pig group as compared to therHSA in JH14 adjuvant control group. The graph illustrates a lack ofenhanced response until after the third injection, and is consistentwith embodiments described herein.

FIG. 7 shows mean pig ELISA titers to vaccine antigen SST from Example10. V shows vaccination. The boxed numbers represent percentseroconversion for the study animals. 100% seroconversion was seen fromweek 4 to week 12.

FIG. 8 shows mean pig ELISA titers to vaccine antigen SST from Example10. V shows vaccination. Significant increase in anti-SST IgG wasdetected at weeks 8 to 12.

FIG. 9 shows a graph of the predicted B-cell epitope characteristicsalong the sequence of the polypeptide conjugate of SEQ ID NO: 13. Thecarrier polypeptide (CAT inactivated) and target antigen (SST) bothexhibit low predicted B-cell epitope scoring; however, the linkerpolypeptide portion exhibits high predicted B-cell epitope scoring.

IDENTIFICATION OF SEQUENCES AND SEQUENCE IDENTIFIERS SEQ ID NO: 1AGCKNFFWKTFTSC SEQ ID NO: 2 (His192→Gly, His193→Gly):atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcattttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttggtggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcagSEQ ID NO: 3: (His192→Gly, His193→Gly):mekkitgyttvdisqwhrkehfeafqsyaqctynqtyqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvggavcdgfhvgrmlnelqq (210 Aas) SEQ ID NO: 4 (His193→Gly)atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcagSEQ ID NO: 5 (1 His193→Ala)atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatgctgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcagSEQ ID NO: 6 (1 His + CAT wt)atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatggtgccgtttgtgatggcttccatgtcggcagaatgcttaatgaactgcagcagSEQ ID NO: 7 (one H→G):mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvhgavcdgfhvgrmlnelqq (210 Aas) SEQ ID NO: 8: (H→A)mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvhaavcdgfhvgrmlnelqq (210 Aas) SEQ ID NO: 9tgggaactgcaccgttctggtccacgcccgcgccctcgcccacgtccggaattcatg SEQ ID NO: 10welhrsgprprprprpefm (19 Aas) SEQ ID NO: 11 welhrsgprprpefm (15 Aas)SEQ ID NO: 12atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttggtggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcagtgggaactgcaccgttctggtccacgcccgcgccctcgcccacgtccggaattcatggccggctgcaagaacttcttttggaaaacctttacgagctgcSEQ ID NO: 13mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqv

avcdgfhvgrmlnelqqwelhrsgprprprprpefmagcknffwktftsc (243 Aas)SEQ ID NO: 14mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqv

avcdgfhvgrmlnelqqwelhrsgprprprprpefmagcknffwktftsc (243 Aas)SEQ ID NO: 15 gctggctgcaagaatttcttctggaagactttcacatcctgt SEQ ID NO: 16welhrsgprprprpefm (17 Aas) SEQ ID NO: 17 welhrsgprprprprprpefm (21 Aas)SEQ ID NO: 18 welhrsgprprprprprprpefm (23 Aas) SEQ ID NO: 19welhrsgprpefm (13 Aas) SEQ ID NO: 20 welhrsgpkpkpkpkpefm (19 Aas)SEQ ID NO: 21 welhrsgpkpkpkpefm (17 Aas) SEQ ID NO: 22welhrsgpkpkpefm (15 Aas) SEQ ID NO: 23 welhrsgpkpefm (13 Aas)SEQ ID NO: 24 welhrsgpkpkpkpkpkpefm (21 Aas) SEQ ID NO: 25welhrsgpkpkpkpkpkpkpefm (23 Aas)SEQ ID NO: 26: (His192→Ala, His193→Ala):mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvaaavcdgfhvgrmlnelqq (210 Aas)SEQ ID NO: 27: (His192→Ala, His193→Gly):mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvagavcdgfhvgrmlnelqq (210 Aas)SEQ ID NO: 28: (His192→Gly, His193→Ala):mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvgaavcdgfhvgrmlnelqq (210 Aas)SEQ ID NO: 29: (His192, His193):mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvhhavcdgfhvgrmlnelqq (210 Aas) SEQ ID NO: 30 (Linker-SST)welhrsgprprprprpefmAGCKNFFWKTFTSC

Additional sequences are provided in the Tables herein.

DETAILED DESCRIPTION OF THE INVENTION

Chloramphenicol Acetyl Transferase (CAT)-Defective Somatostatin FusionProteins are disclosed in U.S. Pat. Nos. 6,316,004, 7,722,881,7,943,143, 8,367,073 and 8,425,914; each of which is incorporated hereinby reference. Although certain aspects of these fusion proteins werepreviously disclosed, certain additional aspects of the fusion proteinshave now been developed. In addition, a model is provided for developingother polypeptide conjugates exhibiting improved immunogenic response toa low B-cell epitope target antigen in a subject.

In some embodiments, methods are provided to improve an immunologicalresponse in a subject to a target antigen, or shorten the time to inducea specific immunological response or effect due to administration of atarget antigen in a subject, comprising administering to the subject apolypeptide conjugate comprising a carrier polypeptide, a linkerpolypeptide and the target antigen, wherein the linker polypeptideexhibits high % predicted linear B-cell epitopes, and wherein thecarrier polypeptide does not stimulate a substantial T-cell response.The method is particularly effective for target antigens exhibiting low% predicted linear B-cell epitopes.

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure.

Definitions:

The term “amino acid” refers to any of the twenty naturally occurringamino acids as well as any modified amino acid sequences. Modificationsmay include natural processes such as posttranslational processing, ormay include chemical modifications which are known in the art.Modifications include but are not limited to: phosphorylation,ubiquitination, acetylation, amidation, glycosylation, covalentattachment of flavin, ADP-ribosylation, cross-linking, iodination,methylation, and the like.

As used herein, the term “epitope” refers to the site on an antigen towhich B and/or T cells respond.

As used herein, the term “B-cell epitope” or “B cell epitope” refers tothe site of an antigen, such as a protein or other molecule, that isspecifically recognized by antibodies (made by B-cells) of the immunesystem. Knowledge of B-cell epitopes may be used in the design ofvaccines and diagnostics tests.

As used herein, the term “improved immunological response” in a subjectfollowing immunization with the polypeptide conjugate of the inventionrefers to one or more of (1) a statistically significant increase inserum anti-target antigen IgG or serum anti-carrier IgG from the subjectobserved after the first or subsequent immunization with the polypeptideconjugate, compared to control receiving immunization with targetantigen and/or carrier without polypeptide conjugation via highpredicted % B-cell epitope linker, (2) a statistically significantincrease or decrease in a specific effect in the subject in response toadministration of the polypeptide conjugate, or (3) a statisticallysignificant faster immunological response compared to a control.

In some embodiments, the specific effect is increased milk production,increased body weight, increased lean body weight, decreased percentbody-fat, or decreased body weight in the subject compared to a controlwithout immunization.

In some embodiments, the improved immunological response is measured asa statistically significant increase or decrease in a specific effectresponse measurement following immunization with the polypeptideconjugate, such as an increase in specific antibody titer, an increasein volume or weight of milk production compared to a control or to thesame individual over time, an increase in overall body weight, anincrease in lean tissue, or a decrease in body weight.

In some embodiments, the improved immunological response or specificeffect is a faster than predicted specific response, wherein theimproved immunological response occurs within less than one day, lessthan two days, less than three days, less than four days, less than fivedays, less than six days, or less than seven days, post vaccinationfollowing the first or subsequent vaccinations with the polypeptideconjugate. In some embodiments, the improved immunological response is aB-cell mediated immunological response.

As used herein, the term “animal” or “subject” or “patient” refers to avertebrate, typically a mammal, in need of the compositions and/ormethods of the present invention, for example, a human, cow, steer,calf, pig, sheep, goat, or horse in need of treatment of one or moredisease states; or a production animal, such as a cow, steer, calf, pig,sheep, goat, in need of improved desirable production characteristics.

As used herein, the terms “polypeptide conjugate”, or “chimericpolypeptide” refer to a molecule comprising a first target antigenpolypeptide covalently attached to a second, heterologous carrierpolypeptide, such that the first and second polypeptides are expressedin frame. In some embodiments, the first and second polypeptides areattached via a linker or linker segment to optimize expression andfunction of the chimeric polypeptide(s) of the invention. Inembodiments, the linker polypeptide comprises one or more B-cellepitopes, which allow it to preferentially stimulate a B-cell mediatedimmune response in the subject. In some embodiments, the polypeptideconjugate is a fusion protein. In other embodiments, the target antigenmay be linked covalently to the linker, and/or the linker may be linkedto the carrier polypeptide, via chemical means known in the art.

The term “carrier polypeptide” refers to a polypeptide selected for usein the polypeptide conjugate used to increase the molecular weight of apolypeptide conjugate comprising a small molecular weight targetantigen, and thus enhance the ability of the subject to raise specificantibodies to the fusion protein and the target antigen. In someembodiments, the carrier polypeptide does not stimulate a substantialT-cell response.

As used herein, the term “linker polypeptide” or “spacer polypeptide”refers to a short polypeptide used to covalently bind the target antigento the carrier polypeptide. The linker polypeptide is selected toexhibit high % predicted linear B-epitopes, and optionally to positionthe target antigen on the surface of the polypeptide conjugate, andenhance stability of the polypeptide conjugate.

As used herein, the term “target antigen” refers to any polypeptideantigen of interest. In some embodiments, the target antigen exhibitslow % predicted linear B-cell epitopes. A low B-cell target epitope ismost likely to benefit from incorporation to a polypeptide conjugate ofthe invention, particularly in terms of increased immunogenicity.

The term “endotoxin” refers to toxins associated in the cell walls ofgram negative bacteria. In some cases the toxins are lipopolysaccharidecomponents of bacterial membranes or components of outer membrane ofgram negative bacteria cell walls.

“Like amino acid,” as used herein, refers to any amino acid thatmodifies the domain or epitope shape so as to render the proteininactive, as defined below. In some embodiments, the “like amino acid”is a non-conservative amino acid substitution. Non-conservative aminoacid substitutions for His include any non-His amino acid. In someembodiments, non-conservative amino acid substitution for His isselected from Gly, Ala, Val, Leu, or Ile; preferably Gly or Ala.Conservative and non-conservative amino acid replacements are defined inBetts et al., 2003, Amino acid properties and consequences ofsubstitutions, Ch. 14, Bioinformatics for Geneticists, John Wiley &Sons, Ltd., pp. 289-316. Note that in the context of certain embodimentsof the invention, like amino acid may refer to all amino acids, as anyconformational change may result in inactivation.

“Modular,” as used herein, refers to the ability of the carriercomposition to generally be paired with any number of potential targetantigens, for example a target antigen comprising a low predicted B-cellepitope score, without substantial modification, while maintaining itsfunctionality.

The terms “protein,” “peptide,” and “polypeptide” are usedinterchangeably to denote an amino acid polymer or a set of two or moreinteracting or bound amino acid polymers.

The term “inactivated” means at least 75%, more typically 80%, 85%, 90%,95% and most typically 96% 97%, 98%, 99%, 100% of an enzyme is renderedincapable of acting functionally in a body or biological system. Assuch, a substantially inactive CAT enzyme is one that has 75%, 80%, 85%,90%, 95% 96%, 97%, 98%, 99% or 100% of its activity removed. (CATactivity can be determined using known functional assays, for examplebinding of n-Butyryl Coenzyme A to radiolabelled chloramphenicol andsubsequent measurement by Liquid Scintillation Counting (LCS); bydetermining the amount of radioactive label transferred from [¹⁴CacetylCoA to chloramphenicol by thin layer chromatography (see MolecularCloning: A Laboratory Model, 3^(rd) ed., J Sambrook and D W Russell,2001. Cold Spring Harbor Press] or other known or like assays).

“Treatment” or “treating” refers to improvement of a subject relative toan untreated subject in a relatively identical situation. Treatment ortreating generally indicates that a desired pharmacological and/orphysiological effect has been achieved using the compositions andmethods of the present invention. Treatment or treating can includeprophylactic treatments. “Vaccine” refers to any composition that canstimulate the vaccinated subject's immune system to produce antibodiesspecific for a target antigen for the purposes described herein.

B-Cell Epitope Prediction

As used herein, the term “B cell epitope” or “B-cell epitope” refers tothe site of a protein or other molecule that is specifically recognizedby antibodies (made by B-cells) of the immune system. Knowledge ofB-cell epitopes may be used in the design of vaccines and diagnosticstests.

Most protein B-cell epitopes are composed of different parts of theprotein chain brought into proximity by the folding of the protein.These are called “discontinuous epitopes”. In about 10% of epitopes, thecorresponding antibodies are cross-reactive with a linear peptideepitope that is a fragment of the protein. These epitopes are called“linear epitopes” or “continuous epitopes” made up of a single stretchof the polypeptide chain (a contiguous amino acid sequence).

Traditionally, a peptide fragment scanning method can be employed toidentify a B-cell epitope; however, due to high cost and timeconstraints, this method has limited practical applicability. Predictionmethods are much more cost effective.

The classical way of predicting linear B-cell epitopes is by the use ofpropensity scale methods. These methods assign a propensity value toevery amino acid, based on their physico-chemical properties.Fluctuations in the sequence of prediction values are reduced byapplying a running average window. Several propensity scale methodsexist, for example, as described by Pellequer at al. Pellequer J,Westhof E, Van Regenmortel M: Predicting the location of continuousepitopes in proteins from their primary structure. Methods Enzymol 1991,203:176-201.

Larsen et al. 2006 describe an improved method for predicting linearB-cell epitopes. Larsen tested a variety of prediction models andreported the best single method for predicting linear B-cell epitopes isthe hidden Markov model. In order to make more accurate predictions,Larsen combined the hidden Markov model with one of the best propensityscale methods to obtain the BepiPred method. BepiPred 1.0 serverpredicts the location of linear B-cell epitopes using a combination of ahidden Markov model and a propensity scale model. Jens Erik PontoppidanLarsen, Ole Lund and Morten Nielsen, “Improved method for predictinglinear B-cell epitopes”, Immunome Research 2:2, 2006. In someembodiments, prediction of BepiPred 1.0 score for a polypeptide can beperformed using an online toolbox, for example, as found athttp://tools.immuneepitope.org/bcell/.

In some embodiments, the % predicted linear B-cell epitopes for apolypeptide of interest, for example, a target antigen, carrierpolypeptide or a linker polypeptide, is calculated by a methodcomprising

-   -   (1) generating a BepiPred 1.0 predicted Linear B-cell epitope        score for each amino acid in the amino acid sequence of the        polypeptide of interest;    -   (2) counting the amino acids in the polypeptide of interest for        which the BepiPred 1.0 predicted Linear B-cell epitope score is        above a threshold value;    -   (3) dividing the number of amino acids above the threshold value        by the total number of amino acids in the polypeptide of        interest to get a fraction; and    -   (4) multiplying the fraction by 100 to get the % predicted        Linear B-cell Epitopes for the polypeptide of interest.

In some embodiments, the threshold value for the % predicted linearB-cell epitopes is selected from −0.2, −0.1, 0, 0.1, 0.2, 0.25, 0.3,0.35, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, or 1.3. In someembodiments, the threshold is selected from 0.1, 0.2, 0.25, 0.3, or0.35. In some embodiments, the threshold is 0.2.

The BepiPred 1.0 method was used herein, as shown in Example 12, topredict B-cell epitopes in a polypeptide conjugate and component targetantigen, carrier polypeptide, and linker polypeptides. Model predictionswere initially based on amino acid sequences for the polypeptideconjugate disclosed in Chloramphenicol Acetyl Transferase(CAT)-Defective Somatostatin Fusion Protein disclosed in U.S. Pat. Nos.7,722,881 and 8,425,914, each of which is herein incorporated herein byreference. A graph of the predicted B-cell epitope characteristics alongthe sequence of the polypeptide conjugate of SEQ ID NO: 13 is shown inFIG. 9 with a threshold of 0.2. The linker portion of the polypeptideconjugate exhibits a high predicted linear B epitope score compared tothe carrier (CATinactivated) and the target antigen (SST), as shown inFIG. 9.

Remarkably, the linker having high % predicted linear B-cell epitopesimparts enhanced B-cell epitope characteristics to the polypeptideconjugate which results in improved immunogenicity of the targetantigen, and improved immunological response/effect in the animalfollowing administration, for example, as shown in improved rapid milkproduction, as shown in FIG. 4.

In one embodiment, a method is provided to enhance the immunogenicity ofa target antigen having low predicted linear B-cell epitope scoring, themethod comprising attaching covalently a carrier polypeptide to thetarget antigen by an intervening linker polypeptide to form apolypeptide conjugate, wherein the linker polypeptide exhibits higherpredicted linear B-cell epitope scoring than the target antigen.

In one aspect, the target antigen is attached to the linker by acovalent bond selected from an amide bond, a disulfide bond, a urethanebond, a carbonate bond, or an ester bond. In one aspect, the carrierpolypeptide is attached to the linker by a covalent bond selected froman amide bond, a disulfide bond, a urethane bond, a carbonate bond, oran ester bond. In one embodiment, the carrier-linker-target antigen is afusion protein, wherein the covalent bond is an amide bond.

In another embodiment, a method is provided to improve theimmunogenicity of a target antigen having low predicted linear B-cellepitope scoring, the method comprising attaching covalently a carrierpolypeptide to the target antigen by an intervening linker polypeptide,wherein the linker polypeptide and/or the carrier polypeptide exhibithigher linear B-cell epitope scoring than the target antigen.

In one embodiment, a method is provided to shorten the time to exhibit aspecific immune response or effect to a target antigen in a subjectfollowing exposure to a vaccine comprising a polypeptide conjugatecomprising a target antigen, comprising administering to the subject apolypeptide conjugate comprising the target antigen, a linkerpolypeptide, and a carrier polypeptide, wherein one or both of thecarrier polypeptide and the linker polypeptide exhibit higher predictedlinear B-cell epitope scoring than the target antigen. In some aspects,the exposure of the subject to the polypeptide conjugate comprising thetarget antigen is the first, second, third, fourth, fifth, sixth, orsubsequent exposure to the target antigen. In some embodiments, thespecific immune response to the target antigen occurs in the subjectwithin a period of six days, five days, four days, three days, two days,one day, or less than one day following exposure to the target antigen.

In one embodiment, a method is provided to shorten the time to exhibit aspecific effect of a target antigen in a subject following exposure tothe target antigen, comprising administering to the subject a fusionprotein comprising the target antigen, a linker polypeptide, and acarrier polypeptide, wherein one or both of the carrier polypeptide andthe linker polypeptide exhibit higher predicted linear B-cell epitopescoring than the target antigen. In some aspects, the exposure of thesubject to the target antigen is the first, second, third, fourth,fifth, sixth, or subsequent exposure to the target antigen. In someembodiments, the specific effect of the target antigen occurs in thesubject within a period of six days, five days, four days, three days,two days, one day, or less than one day following exposure to the targetantigen.

Target Antigens:

In some embodiments, the target antigen is selected from any polypeptideantigen of interest, or peptide fragment thereof. In some embodiments,the target antigen or fragment thereof is selected from a peptidefragment of from 5 to 40 amino acids in length. In some embodiments, thetarget antigen, or fragment thereof, is from 8 to 35 amino acids inlength. In some embodiments, the target antigen is from 10 to 25 aminoacids in length. In some embodiments, the target antigen, or fragmentthereof, is selected from a peptide length that is 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids in length.

In some embodiments, the target antigen exhibits low % predicted linearB-cell epitopes. A low B-cell target epitope is most likely to benefitfrom incorporation to a polypeptide conjugate of the invention,particularly in terms of increased immunogenicity.

In some embodiments, the target antigen or fragment thereof exhibits low% predicted Linear B-cell Epitopes as calculated by the BepiPred 1.0method. In some embodiments, the target antigen or fragment thereofexhibits <10%, <7%, <5%, <4%, <3%, <2%, <1%, <0.1% or 0% predictedLinear B-cell Epitopes wherein the % predicted linear B-cell epitopes isdetermined by BepiPred 1.0 scoring of the target antigen amino acidsequence. Briefly, the amino acid sequence for the target antigen isdetermined by Bepipred 1.0 scoring as provided herein. The amino acidsscored above a threshold value are counted, and divided by the totalnumber of amino acids in the target antigen to get a fractional value.The fractional value is multiplied by 100 to get a % predicted linearB-cell epitope value for the target antigen.

In some embodiments, the target antigen is selected from GonadotropinReleasing Hormone, Luteinizing Hormone Releasing factor, Calcitoninneuropeptides, Myostatin, HIV envelope protein, Outer membrane Protein(OMP) from pathogenic bacteria (TB. N. meningitis); or a target antigenfragment thereof of from 5 to 40 amino acids in length.

In some embodiments, the target antigen is selected from somatostatin14, a fragment of Mycobacterium tuberculosis outer membrane protein A(OmpATb), Gonadotropin Releasing Hormone (GnRH1), a Calcitoninneuropeptide fragment, a myostatin fragment (growth differentiationfactor 8; GDF8), a Human Immunodeficiency Virus envelope glycoproteinpolypeptide fragment, or a fragment of Neisseria meningitides outermembrane protein complex (e.g., Omp85).

In some embodiments, the target antigen is selected from a peptide from5 amino acids to 40 amino acids in length.

In some embodiments, the target antigen is Somatostatin 14 (SEQ ID NO:1).

In some embodiments, the target antigen is not Somatostatin 14 (SEQ IDNO: 1).

In some embodiments, the target antigen comprises a fragment ofMycobacterium tuberculosis outer membrane protein A (OmpATb).Mycobacterium tuberculosis outer membrane protein A (OmpATb) has twofunctions: as a pore-forming protein with properties of a porin, and inenabling M. tuberculosis to respond to reduced environmental pH. TheN-terminal domain of OMPATb is required for membrane translocation andpore-forming activity in Mycobacteria. Alahari et al., J Bacteriology2007, 189(17): 6351-6358. In some embodiments, the target antigen is anOmpATb polypeptide. The amino acid sequence of M. tuberculosis H37Rv(GB:AL123456): Rv0899—outer membrane protein A (OmpA Tb) is shown at SEQID NO: 31. In some embodiments, the target antigen is selected from afragment of OmpA Tb exhibiting low % predicted linear B-cell epitopes.In some embodiments, the target antigen is an N-terminal fragment ofOmpATb. In some embodiments, the target antigen is selected from one ormore of OmpATb fragments OmpATb 32-48 (SEQ ID NO: 32), 75-86 (SEQ ID NO:33), 104-134 (SEQ ID NO: 34), 235-247 (SEQ ID NO: 35), or 271-289 (SEQID NO: 36), 294-302 (SEQ ID NO: 37), or 319-326 (SEQ ID NO: 38), or afragment thereof.

In some embodiments, the target antigen comprises an amino acid sequenceaccording to Gonadotropin Releasing Hormone (GnRH1), or a fragmentthereof. GnRH1 is also known as luteinizing hormone releasing factor,controls a complex process of follicular growth, ovulation, and corpusluteum maintenance in the female, and spermatogenesis in the male. GNRHantagonists block the effects of GnRH and have applications in prostatecancer, fertility treatment, breast cancer, endometriosis, and may beuseful in benign prostatic hyperplasia, and as potential contraceptiveagents. It is believed that a polypeptide complex will be useful forsimilar applications. In some embodiments, the target antigen comprisesthe amino acid sequence of GnRH1, or EHWSYGLRPG, or a fragment thereof.In some embodiments, the target antigen is selected from one or more ofGnRH1 fragments GnRH1 1-8 (SEQ ID NO: 40), or GnRH1 1-9 (SEQ ID NO: 41).

In some embodiments, the target antigen comprises an amino acid sequenceselected from calcitonin neuropeptide, or a fragment thereof, orpreprocalcitonin, or a fragment thereof. Preprocalcitonin is about139-141 amino acids in length and may exist in more than one isoform. Ina specific embodiment, the target antigen has an amino acid sequencecorresponding to Calcitonin 85-105, or CGNLSTCMLGTYTQDFNKFHT (SEQ ID NO:43), a 21 amino acid fragment which exhibits only 4.8% predicted %linear B-cell epitopes.

In some embodiments, the target antigen comprises an amino acid sequenceselected from a low B-cell epitope fragment of a member of thecalcitonin-like family selected from calcitonin, Calcitinin-Gene RelatedPeptide, or calcitonin neuropeptide. Calcitonin is a 32 amino acidpolypeptide hormone that acts to reduce blood calcium. Calcitonin isused therapeutically for the treatment of osteoporosis. Alternativesplicing of the gene coding for calcitonin produces a related peptidecalled Calcitonin-Gene-Related Peptide (CGRP) is a 37 amino acidvasoactive neuropeptide that is widely distributed in central andperipheral neurons. Ma, Nature and Science 2(3) 2004, 41-47. In humans,two types of CGRP exist-alpha and beta, which differ in sequence by afew amino acids. CGRP induces vasodilatation in a variety of vessels,including the coronary, cerebral and systemic vasculature. CGRPantagonists are being studied for use in treating migraines.

In some embodiments, the target antigen comprises an amino acid sequenceselected from a fragment of calcitonin neuropeptide or preprocalcitonin.Preprocalcitonin is about 139-141 amino acids in length and may exist inmore than one isoform, for example, as shown in SEQ ID NO: 42. In aspecific embodiment, the target antigen has an amino acid sequencecorresponding to Calcitonin 85-105, or CGNLSTCMLGTYTQDFNKFHT (SEQ ID NO:43), a 21 amino acid fragment which exhibits only 4.8% predicted %linear B-cell epitopes. Minvielle et al., JBC 266(36) 24627-31 (1991).

In some embodiments, the target antigen comprises an amino acid sequenceselected from an amino acid sequence of myostatin, or a fragmentthereof. Myostatin (also known as growth differentiation factor 8; GDF8)is a secreted growth differentiation factor. Animal slacking myostatinhave significantly larger muscles. Blocking the activity of myostatinmay be of therapeutic benefit in muscle wasting diseases such asmuscular dystrophy. Gonzalez-Cavadid et al., 1998, PNAS USA 95, pp.14938-14943. In some embodiments, the target antigen comprises an aminoacid sequence selected from a myostatin fragment selected from 1-20 (SEQID NO: 46), 52-68 (SEQ ID NO: 47), 136-162 (SEQ ID NO: 48), 169-179 (SEQID NO: 49), 204-216 (SEQ ID NO: 50), 280-302 (SEQ ID NO: 51), 311-329(SEQ ID NO: 52).

In some embodiments, the target antigen comprises an amino acid sequenceselected from a fragment of Human Immunodeficiency Virus envelopeglycoprotein amino acid sequence, wherein the fragment exhibits low %predicted linear B-cell epitopes. For example, gp 120 plays a role inthe ability of HIV-1 to enter CD4+ cells. Unfortunately, neutralizingantibody responses drive the evolution of HIV-1 envelope during recentinfection, therefore additional vaccine constructs are desirable. Frostet al., 2005, PNAS 102(51):18514-18519.

In some embodiments, the target antigen comprises an amino acid sequenceselected from a fragment of Neisseria meningitides outer membraneprotein complex (e.g., Omp85). Omp85 is required for outer membraneprotein assembly in gram-negative bacteria and in mitochondria.Volokhina et al., 2009, J Bacteriol, 191(22): 7074-7085.

Table 1 shows % predicted linear B-cell epitopes for certain targetantigens and fragments thereof.

TABLE 1Target Antigens, Fragments and % predicted linear B-cell Epitopes*BepiPred 1.0 Score- No. Aas % above Predicted threshold linear B-Target Antigen and Fragment (threshold Total cell Sequences value = 0.2)No. Aas Epitopes Name AGCKNFFWKTFTSC (SEQ ID 0 14 0% Somato NO: 1)statin 14 MASKAGLGQTPATTDARRTQKF 194 326 60% M. YRGSPGRPW

tuberculosis -

AFERPQSVTGPTGVLPTLTPT outer STRGA

SGNTVTL membrane IGDFPDEAAK

protein A

FSSA (Omp ATb) EPVFTASVPIPDFGLKVERDTVT LTGTAPSSEHKDAVKRAATSTWPDMKIVNNIEVTGQAPPGPPASG PCADLQSAINAVTGGPIAFGNDG ASLIP

KLKACPD ARVTINGYTDNTGSEGIN

VAGD

NPIASNATPEGRAKN

 (SEQ ID NO: 31)

  (SEQ ID 0 17 0% OmpA NO: 32) Tb 32- 48

 (SEQ ID NO: 33) 0 12 0% OmpA Tb 75- 86

0 31 0% OmpA

 (SEQ ID NO: 34) Tb 104- 134

 (SEQ ID NO: 35) 0 11 0% OmpA Tb 235- 246

 (SEQ 0 19 0% OmpA ID NO: 36) Tb 271- 289

 (SEQ ID NO: 37) 0 10 0% OmpA Tb294- 302

 (SEQ ID NO: 38) 0 10 0% OmpA Tb 319- 326pyroGlu-His-Trp-Ser-Tyr-Gly-Leu- 6 10 60% GnRH Arg-Pro-Gly-NH2 1-10EHWSYGLRPG (SEQ ID NO: 39) EHWSYGLR (SEQ ID NO: 40) 0 8 0% GnRH1 1-8EHWSYGLRP (SEQ ID NO: 41) 1 9 11% GnRH1 1-9 MGFQKFSPFLALSILVLLQAGSL 72139 52% calcitonin HAAPFRSALESSPAPATLSEDEA neuropeptide-RLLLAALVQNYVQMKASELEQ preprocalcitonin EQEREGSSLDSPRSKRCGNLSTCMLGTYTQDFNKFHTFPQTAIGV GAPGKKRDMSSDLERDHRPHVS MPQNA (SEQ ID NO: 42)CGNLSTCMLGTYTQDFNKFHT 1 21 4.8% Calcitonin (SEQ ID NO: 43) 85-105 AARSKRCGNLSTCMLGTYTQDFNK 17 36 47% Calcitonin FHTFPQTAIGVGAP (SEQ ID NO:AA 44) 81-116

vdlnenseqkenv 161 375 43% myostatin ekeglcnactwrqntkss

a pniskdvirqllpkapplrelidqydvqrddssd gsledddyhattetiitmptesdflmqvdgkp

etpt

kdgtrytgirslkldmnpgtgiw

kqpesnlgieikaldenghdlavt fpgpgedglnpflevkvtdtpkrsrrdfgldcde hstes

rykany cs

anprgsagpcct ptkmspinmlyfngkeqiiygkipamvvdrcgcs (375 Aas) (SEQ ID NO: 45)

 (SEQ ID NO: 0 20 0% Myostatin 46) 1-20

 (SEQ ID NO: 47) 0 17 0% Myostatin 52-68

 (SEQ 0 28 0% Myostatin ID NO: 48) 136-162

 (SEQ ID NO: 49) 0 13 0% Myostatin 169-179

 (SEQ ID NO: 50) 0 13 0% Myostatin 204-216

 (SEQ ID 0 23 0% Myostatin NO: 51) 280-302

 (SEQ ID NO: 0 19 0% Myostatin 52) 311-329 Note: regions of lowpredicted linear B cell epitope characteristics are shown in italics insome target antigens and fragments in Table 1.

In some embodiments, the target antigen in the carrier-linker-targetantigen fusion protein is not somatostatin-14. In other embodiments, thetarget antigen in the carrier-linker-target antigen fusion proteincomprises the somatostatin-14 amino acid sequence of SEQ ID NO: 1.

In some embodiments, the target antigen is Somatostatin-14 (SST). SST isknown to have strong inhibitory effect on a large number of hormonesinvolved in the growth and utilization of food in animals. As previouslydescribed, for example, in U.S. Pat. No. 7,722,881, chimeric versions ofsomatostatin were used for immunization of animals that subsequentlyexhibited an increase in daily weight gain or an increase in milkproduction.

Linker Polypeptides

Linker polypeptides according to the invention are selected or designedby determining a high B cell epitope score for the peptide. In someembodiments, the B cell epitope scoring system is BepiPred 1.0, asprovided herein. In some embodiments, the linker polypeptide exhibitshigher predicted linear B-cell epitope scoring than the target antigen.

In some embodiments, the linker polypeptideexhibits >50%, >60%, >70%, >80%, >90%, >95% or >99% predicted linearB-cell epitopes, wherein the % predicted linear B-cell epitopes isdetermined by (1) generating a BepiPred 1.0 predicted Linear B-cellepitope score for the amino acid sequence of the linker polypeptide, (2)dividing the Bepiprep 1.0 predicted Linear B-cell epitope score by thenumber of amino acids in the linker polypeptide and (3) multiplying theresulting number by 100 to get the % predicted Linear B-cell Epitopes,as provided herein. In some embodiments, the linker polypeptideexhibits >80%, >90% or >95% predicted linear B-cell epitopes.

In some embodiments, the linker polypeptide is a peptide of 5 to 35amino acids in length, 8 to 30 amino acids in length or 10 to 25 aminoacids in length.

In some embodiments, the linker polypeptide is selected from B cellepitope known in the art. In some embodiments, the linker polypeptidecomprises an amino acid sequence selected from linker polypeptideselected from SEQ ID NO: 10, 11, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,53, 54, 55, 56 or 57. In some embodiments, the linker polypeptidecomprises an amino acid sequence that exhibits 90% or greater, or 95% orgreater, sequence identity with an amino acid sequence selected from SEQID NO: 10, 11, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 53, 54, 55, 56 or57.

Example linker polypeptides and linear B cell epitope scoring are shownin Table 2.

TABLE 2 Linker Polypeptides with High Linear B Cell Epitope ScoringBepiPred 1.0 SEQ Score*-No. Aas % Predicted ID Linker Polypeptideabove threshold Total linear B-cell NO: Sequence of 0.2 No. Aas EpitopesSource 10 welhrsgprprprprpefm 16 19 84% U.S. Pat. Nos. 7,722,881;8,425,914; or 2013/0149332 11 welhrsgprprpefm 12 15 80% U.S. Pat. Nos.7,722,881; 8,425,914; or 2013/0149332 16 welhrsgprprprpefm 14 17 82%U.S. Pat. Nos. 7,722,881; 8,425,914; or 2013/0149332 17welhrsgprprprprprpefm 18 21 86% U.S. Pat. Nos. 7,722,881; 8,425,914; or2013/0149332 18 welhrsgprprprprprprpefm 20 23 87% U.S. Pat. Nos.7,722,881; 8,425,914; or 2013/0149332 19 welhrsgprpefm 10 13 77%U.S. Pat. Nos. 7,722,881; 8,425,914; or 2013/0149332 20welhrsgpkpkpkpkpefm 16 19 84% U.S. Pat. Nos. 7,722,881; 8,425,914; or2013/0149332 21 welhrsgpkpkpkpefm 14 17 82% U.S. Pat. Nos. 7,722,881;8,425,914; or 2013/0149332 22 welhrsgpkpkpefm 12 15 80% U.S. Pat. Nos.7,722,881; 8,425,914; or 2013/0149332 23 welhrsgpkpefm 10 13 77%U.S. Pat. Nos. 7,722,881; 8,425,914; or 2013/0149332 24welhrsgpkpkpkpkpkpefm 18 21 86% U.S. Pat. Nos. 7,722,881; 8,425,914; or2013/0149332 25 welhrsgpkpkpkpkpkpkpe 20 23 87% U.S. Pat. Nos. fm7,722,881; 8,425,914; or 2013/0149332 53 PPKDTNQTQPATQPA 15 15 100%Etz et al., 2002, PNAS 99(10): 6673; FIG. 5, Staphylococcus aureussynthetic peptide B-cell epitope (LPX- TGp5 epitope) 54 QGPGAPQGPGAPQGP18 18 100% Plasmodium GAP yoelii circumsporozoite (CS) proteinB cell epitope (PyCS-B epitope3) Shiratsuchi et al., 2010, J. Clin.Investigation 120(10): 3688- 3701 55 QGPGAPQGPGAP 12 12 100% PyCS-Bepitope2 56 QGPGAPQGPGAPQGP 24 24 100% PyCS-B GAPQGPGAP epitope4 57EHKYSWKS 8 8 100% Dengue Virus Type 1 B cell epitope; (DEN- 1 peptide)Wu et al., 2001 J. Clin. Microbiol. 39(3): 977-982*http://www.cbs.dtu.dk/services/BepiPred/333

Linker embodiments, therefore, were previously optimized in length andcomposition to ensure CAT-defective recombinant protein expression invarious microorganisms, and in particular in E. coli, as disclosed infor example, in U.S. Pat. No. 7,722,881. Original constructs asdescribed in U.S. Pat. No. 6,316,004, included a linker having rare E.coli codons and required the co-expression of rare tRNAs from a secondor helper plasmid. Linker embodiments disclosed in co-pending U.S.Patent Publication No. 2013/0149332, hereby incorporated by reference inits entirety, removed these rare E. coli codons and the need for asecond helper plasmid, an improvement over previous technology.

As provided herein, the linkers of the present invention are now shownto offer further improvement by providing optimal B epitope bindingsites, allowing the fusion proteins of the invention to directlystimulate B cells, which represents a large inventive leap over previouscompositions.

In some embodiments, the linker polypeptide provided herein comprises anamino acid sequence repeat of (Arg-Pro)_(n) or (Lys-Pro)_(n), where nrepresents an integer, preferably from 2 to 6. In some aspects, thelinker polypeptide is selected from one of SEQ ID NOs: 10, 11, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 53, 54, 55, 56 or 57. In a specificaspect, n=4 and the linker polypeptide is SEQ ID NO: 10.

As shown in FIGS. 1 and 2, repeated Pro-Arg sequences provide a highnumber of B epitopes. Other possible linker sequences are disclosed inU.S. Patent Publication No. 2013/0149332. In some embodiments, thepresent linker also directly stimulates B cells, providing for noveldrug-like characteristics. In a particular embodiment, for example, thecombination of somatostatin-14 attached to a substantially inactivatedCAT enzyme (histidine replaced constructs) by a linker with a greaternumber of B epitopes showed unexpected and surprising improvement overother materials when used to immunize target animals for enhancedproductivity.

In some embodiments, a method is provided for enhancing an immunogenicresponse to a target antigen in a subject, the method comprisingadministering a polypeptide conjugate comprising the target antigen, alinker polypeptide, and a carrier polypeptide, wherein the linkerpolypeptide exhibits >50%, >60%, >70% or preferably >80% predictedlinear B-cell epitopes, wherein the % predicted linear B-cell epitopesis determined by (1) generating a BepiPred 1.0 predicted Linear B-cellepitope score for the amino acid sequence of the linker polypeptide, (2)dividing the Bepiprep 1.0 predicted Linear B-cell epitope score by thenumber of amino acids in the linker polypeptide and (3) multiplying theresulting number by 100 to get the % predicted Linear B-cell Epitopes.

In some embodiments, the polypeptide conjugate comprises, fromN-terminus to C-terminus, a carrier polypeptide comprising aninactivated chloramphenicol acetyltransferase (CAT) selected from SEQ IDNO: 3, 7, 8, 26, 27, 28, or 29; a linker polypeptide selected from SEQID NO: 10, 11, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 53, 54, 55, 56 or57; and target antigen selected from SEQ ID NO: 1, 32, 33, 34, 35, 36,37, 38, 40, 41, 43, 44, 46, 47, 48, 49, 50, 51, or 52.

In some embodiments, the carrier polypeptide does not stimulate asubstantial T cell mediated response. In some embodiments, the T-cellresponse is determined by a T cell epitope prediction program. In someembodiments, the T-cell epitope prediction can be performed by anymethod known in the art. For example, as disclosed in U.S. Pat. No.8,121,797, which is incorporated herein by reference. Any computationalbased procedure for T-cell epitope may be employed. In some embodiments,the T cell epitope prediction is performed by a method selected from:Artificial neural network (ANN) Neilsen, M. et al., Reliable predictionof T-cell epitopes using neural networks with novel sequencerepresentations; Schuler M M et al., SYFPEITHI: database for searchingand T-cell epitope prediction. Methods Mol Biol. 2007; 409:75-93;Stabilized Matrix Method (SMM) Peters, B. et al., Generatingquantitative models describing the sequence specificity of biologicalprocesses with the stabilized matrix method, BMC Bioinformatics, 2005,May 31; 6:132; SMM with a Peptide:MHC Binding Energy Covariance matrix(SMMPMBEC) Kim, Y. et al., Derivation of an amino acid similarity matrixfor peptide: MHC binding and its application as a Bayesian prior, BMCBioinformatics 2009 Nov. 30; 10:394; and NetMHCpan Hoof, I. et al.,NetMHCpan, a method for MHC class I binding prediction beyond humans,Immunogenetics, 2009 January; 61(1):1-13, each of which is incorporatedherein by reference. In some embodiments, the T cell epitope predictionprogram version is selected from NetMHC 2.8 or NetMHC(ANN) 3.4, forexample, as found athttp://tools.immuneepitope.org/main/html/tcell_tools.html

In another embodiment, novel adjuvant compositions are provided for usein the treatment of patients having one or more diseases or conditionsdescribed herein.

In one particular embodiment, the polypeptide conjugate comprises atarget antigen that is a somatostatin-based antigen attached to thecarrier embodiments via the linker described herein and is used in thetreatment of growth hormone or insulin-like growth factor 1 deficiencyrelated disease states or conditions, e.g., growth deficiency inchildren, growth deficiency in adults, lack of adequate endogenousgrowth hormone secretions, healing of burns, obesity, cardiac disease,etc. In some embodiments, the vaccines described herein are designed foroptimal use in vertebrates, particularly humans, and provide forenhanced primary immunogenicity with the absence or limitation of ananamnestic response. Conjugated vaccine embodiments herein are usefulwith other antigen combinations beyond those useful in the treatment ofa target disease state, and may be combined with any suitable targetantigen (in the absence of a carrier). Additional novel stand-aloneembodiments are therefore within the scope of the present invention.

With regard to somatostatin, vaccines are provided that result inimmunogenicity against somatostatin that results in diminution ofsomatostatin and thereby removal of a proportion of the inhibition thatsomatostatin exerts on growth hormone release and thereby insulin-likegrowth factor 1 release. Vaccine embodiments herein are optimized forboth safety and function, having highly immunogenic somatostatinconstructs in safe and highly effective adjuvant compositions. Vaccinesof the present invention require relatively smaller amounts of antigen(as compared to conventional vaccines), have enhanced storage life, andare lower cost. In some embodiments, somatostatin-based vaccines of theinvention do not cause an anamnestic response, and may be administeredroutinely and with increased safety.

In some embodiments, the carrier is selected from a protein selectedfrom a bacterial chloramphenicol acetyl transferase (CAT) polypeptide,an inactivated chloramphenicol acetyl transferase (CAT) polypeptide witha C-terminal deletion, an inactivated chloramphenicol acetyl transferase(CAT) with one or more amino acid substitutions, a beta-galactosidase, adihydrofolate reductase, or a hydrophobic synthetic polypeptide. In someembodiments, the carrier polypeptide is selected from an inactivatedchloramphenicol acetyl transferase (CAT) or a dihydrofolate reductase(DHFR). In some embodiments, the carrier polypeptide does not stimulatea substantial T-cell response.

In some embodiments, the carrier polypeptide is a chloramphenicol acetyltransferase (CAT) polypeptide. CAT is a bacterial enzyme that detoxifiesthe antibiotic chloramphenicol and is responsible for chloramphenicol inbacteria. The enzyme attaches an acetyl group from acetyl co-A tochloramphenicol which prevents chloramphenicol from binding toribosomes. A histidine residue located in the C-terminal portion of theenzyme is said to be responsible for the catalytic mechanism. In someembodiments, the carrier polypeptide is a CAT enzyme sequence.

In some embodiments, the carrier polypeptide is a CAT enzyme that is atleast partially inactivated by truncation of the polypeptide at theC-terminus and or replacement of one or both of the His residues in thecatalytic site. In some aspects, the CAT enzyme is at least partiallyinactivated by replacement of the His residues at position 192, 193 inthe catalytic site of the enzyme.

In some embodiments, the carrier polypeptide is an inactivatedchloramphenicol acetyl transferase (CAT) polypeptide selected from SEQID NOs: 3, 7, 8, 26, 27, 28 or 29. In some embodiments, the carrierpolypeptide is SEQ ID NO: 3.

Novel Vaccine Embodiments for Use in Treatment of Disease

The methods of the invention include the use of a vaccine comprising apolypeptide conjugate such as a fusion protein comprising a targetantigen conjugated to a carrier polypeptide that is an inactivated CATenzyme via a linker that exhibits higher predicted linear B-cell epitopescoring than the target antigen. The carrier is attached to a targetantigen via a linker, and the polypeptide conjugate is capable ofgenerating an immune response against the target antigen and similarproteins, while directly targeting B cell epitopes. In some embodiments,neither the target antigen, the linker, nor the carrier polypeptide,stimulate a substantial T-cell response. Administration of thepolypeptide conjugate such as a fusion protein to a subject results in adesirable B-cell mediated immune response against the carrier, linkerand target antigen, but avoids the development of T-cell memory,limiting recognition and neutralization of the construct by the immunesystem upon subsequent administrations.

In further embodiments, vaccines comprising the polypeptide conjugateare utilized in a secondary or booster vaccination in a prime-boostvaccine. Using the compositions of the invention as a secondary orbooster vaccination has the advantage of allowing further beneficialtreatment of a disease state by generating an immune response, withoutgenerating a significant T-cell mediated response.

In some embodiments, the target antigens are recombinant proteinstypically having truncated or modified sequences, allowing attachment tothe linker while maintaining the immunogenicity of the unmodifiedprotein. For example, a protein may be shortened by one or more C- orN-terminal amino acids to allow attachment to the linker.

In some embodiments, the carrier polypeptide is an inactivated CATprovided by the following nucleic acid constructs that encode aninactive CAT enzyme that is without 10 C-terminal amino acids. In someembodiments, the carrier polypeptide CAT construct comprises one or twohistidine amino acid modifications therein which result in partial tocomplete CAT inactivation. In one embodiment, the CAT enzyme isinactivated by removing the imidazole group of His193 (His195 in thecanonical CAM_(III) variant) and replacing with a non-conservative aminoacid replacement. In another embodiment the CAT enzyme is inactivated byremoving the imidazole groups of both His 193 and the nearby His192(respectively His195 and His194 for CAT_(III) and replacing with anon-conservative amino acid replacement. Removal of the essential His193(His195 in CATO imidazole group from the active site of CAT andreplacement with an alanine, glycine or other non-conservative aminoacid results in substantial inactivation of the CAT enzyme (see forexample, Lewendon A et al. (1994) Replacement of catalytic histidine-195of chloramphenicol acetyl transferase: evidence for a general base rolefor glutamate. Biochemistry. 33(7):1944-50; White et al., (2000)Characterization of Chloramphenicol and Florfenicol Resistance inEscherichia coli associated with Bovine Diarrhea. J. Clin. Micro 38(12)p 4593-4598, each of which is incorporated by reference herein for allpurposes). In some embodiments, the carrier peptide is an inactivatedCAT enzyme comprising a removal of the imidazole group of His192 alone(His 194 for CAT₁₁₁) and replacement with an alanine, glycine or othernon-conservative amino acid substitution.

In some aspects, the one or more replaced histidine amino acids areencoded by nucleic acids located at position numbers 574-576 and 577-579of SEQ ID NO: 2 (corresponding to amino acid numbers 192 and 193 in SEQID NO: 3). In some embodiments the nucleic acid sequences of theinvention include SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Chimericproteins of the invention that include the histidine replaced constructsherein provide highly immunogenic proteins with little or no CATactivity. The inactivated CAT enzyme embodiments are attached to atarget antigen. This attachment is made via linker embodiments, asdescribed more fully below.

CAT inactivation, at sites his192 and/or his193, can be accomplished viaany number of known procedures to those skilled in the art includingsite-directed mutagenesis and synthetic gene assembly. In oneembodiment, the nucleic acid sequence that encodes histidine 192 orhistidine 193 are modified to encode an alanine, glycine or other likeamino acid. In another embodiment, the nucleic acid sequences thatencode both histidine 192 and 193 are modified to encode alanine,glycine or other like amino acids. In some embodiments, the carrierpolypeptide is an inactivated CAT enzyme comprising amino acidsubstitutions for both the 192 and 193 histidines selected from thegroup consisting of alanine, alanine; alanine, glycine; glycine,alanine; and glycine, glycine, at the 192 and 193 position,respectively.

In some embodiments, the carrier polypeptide is a CAT deficientpolypeptides, having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 3, 7, and 8 (corresponding to his→gly at both192 and 193, his→gly at 193, and his→ala at 193).

The realization that the inactivated CAT enzyme carrier and linkerscould present various target antigens such as recombinant proteins inthe treatment of diseases or conditions herein without creating ananamnestic response was an unexpected finding of the inventors. Previousvaccine carriers and linkers trigger a normal immune responsecharacterized by T cell response, processing, and a resultingimmunological memory for a vaccine. This response may result inneutralization upon subsequent vaccinations, limiting the safety andeffectiveness of future administrations of the vaccine. As such, therealization, development, and use of the B-epitope containing carrierand/or linkers of the invention, for use with target antigens exhibitinglow B-epitope characteristic, with a reduced anamnestic response, isdesired, representing a significant improvement over existing vaccines.

Note that these “carrier” related improvements of CAT for use with smallmolecules are discussed in co-pending and related U.S. PatentApplication S/N PCT/US08/68195 as well as in U.S. Pat. No. 6,316,004both of which are incorporated by reference herein for all purposes. Inparticular, the inventors herein unexpectedly found that an inactivatedCAT enzyme as a carrier protein for low B epitope-containing targetantigens such as recombinant proteins could avoid the significant healthrisks associated with the enzyme while utilizing the chimeric proteinsenhanced capacity for immunogenicity, resistance to enzyme degradation,increased half-life and enhanced uptake by the patient's macrophages.

In some embodiments, a polypeptide conjugate is provided comprising asubstantially inactivated CAT enzyme covalently attached to a targetantigen via a linker, wherein the linker allows for presentation of thetarget antigen on a global surface of the fusion protein. Linkerembodiments also provide for optimal protease resistance and for optimalepitope exposure. Inclusion of the linker in polypeptide conjugates hasresulted in unexpected improvement over constructs not having the linkersequence(s) of the present invention.

In some embodiments, the immunogenic response to a target antigen is aB-cell mediated immunogenic response.

In some embodiments, the polypeptide conjugate is a fusion protein.

See Table 2, showing % predicted Linear B-cell Epitopes for linkerpolypeptides. Surprisingly, it has been found that the linker of SEQ IDNO: 3 exhibiting >50%, >60%, >70% or preferably >80% predicted LinearB-cell Epitopes imparts the polypeptide conjugate with improvedimmunological response in a subject.

Further, these chimeric constructs show enhanced storage stability ascompared to a target antigen recombinant protein alone. In addition, thepolypeptide conjugates of the present invention provide for greaterhalf-life in the patient of the target antigen given the enhancedresistance to degradation in these materials, especially as compared torecombinant proteins with KLH, tetanus toxoids or CRM.

Embodiments of the invention also provide novel adjuvant compositionsfor enhanced induction of humoral immunity in a target patient. Theseadjuvant compositions provide a significant improvement overconventional materials for the induction of a humoral response and aresafe for use in human targets. Adjuvant compositions herein are usedwith recombinant protein-based antigens to produce vaccines of theinvention. Vaccines of the invention are then useful in the treatment ofnumerous diseases or conditions.

In embodiments herein, all components of adjuvant compositions are ofnon-animal origin, thereby eliminating potential cross-contamination ofvaccinated humans from potentially contaminated adjuvant components. Forexample, embodiments herein can utilize animal origin free Tween 80.Surprisingly, animal origin free Tween 80 shows significantly betterresults in the use of vaccines herein as compared to animal origin Tween80, and eliminates the possibility of animal-based contamination intothe vaccine, e.g., Bovine Spongiform Encephalopathy (BSE). In addition,animal origin free Tween 80 shows better capacity to emulsify ascompared to animal origin Tween 80, providing an additional unexpectedbenefit for its use in accordance to embodiments herein.

Adjuvant embodiments herein are also free of benzene and other likecarcinogenic compounds. These embodiments provide a safety benefit notavailable in most conventional adjuvant compounds. For example,embodiments herein utilize Carbopol 974P or benzene free polycyclicacid.

In one embodiment, the immunologic adjuvant comprises a carbopol base, asqualene base and an arabinogalactan solution. In more detail, theCarbopol base is prepared using Carbopol 974P in water or saline. Thesqualene base is prepared from a combination of squalene, non-animalorigin Tween 80 and Span 85. In some embodiments the squalene base isMF59 (Chiron Corp., Emeryville, Calif.). The arabinogalactan isdissolved in PBS or saline. Adjuvant compositions are combined withchimeric polypeptides of the invention to produce vaccines of theinvention.

In yet another embodiment, the immunologic adjuvant comprises a Carbopolbase, a squalene base and a tragacanthin solution. In more detail, theCarbopol base is prepared using Carbopol 974 P in water or saline. Thesqualene base is prepared from a combination of squalene, non-animalorigin Tween 80 and Span 85. Purified tragacanthin is dissolved in PBSor saline. Adjuvant compositions are combined with chimeric polypeptidesof the invention to produce vaccines or the invention.

Specific adjuvant combination and concentrations are shown in theexamples below. Adjuvants in accordance with the present invention aresafe and effective for human use, avoid animal products, avoid petroleumbased hydrocarbons, and avoid carcinogenic compounds.

Vectors and Host Cells

The present invention also relates to vectors comprising thepolynucleotide molecules of the invention, as well as host cellstransformed with such vectors. Any of the polynucleotide molecules ofthe invention may be joined to a vector, which generally include aselectable marker and origin of replication, for the propagation host ofinterest. Host cells are genetically engineered to include these vectorsand thereby express the polypeptides of the invention. Generally,vectors herein include polynucleotides molecules of the inventionoperably linked to suitable transcriptional or translational regulatorysequences, such as those for microbial or viral host cells. Examples ofregulatory sequences include transcriptional promoters, operators, orenhancers, mRNA ribosomal binding sites, and appropriate sequences whichcontrol transcription and translation. Nucleotide sequences are operablylinked when the regulatory sequences herein functionally relate to thechimeric polypeptide encoding polynucleotides of the invention.

Typical vehicles include plasmids, yeast shuttle vectors, baculovirus,inactivated adenovirus, and the like. In one embodiment the vehicle is amodified pET30b CatSom plasmid. Target host cells for use herein includebacterial host, e.g., E. coli, yeast, SF-9 insect cells, mammaliancells, plant cells, and the like.

In one embodiment, the regulatory sequences include a T7lac, CAT, Trp,or T5 promoter for expression of the chimeric polypeptides of theinvention in E. coli or other like microbes. These regulatory sequencesare known in the art and are used under appropriate and knownconditions.

Where genetically modified green plant cells are utilized forexpression, systems as developed by Planet Biotechnology and others canbe utilized.

Various plasmids of the invention have been constructed for expressionof chimeric polypeptides of the invention through utilization of targetregulatory sequences. Illustrative plasmids can include a T7lacpromoter.

Host cells for expression of target chimeric polypeptides includeprokaryotes, yeast and higher eukaryotic cells. Illustrative prokaryotichosts include bacteria of the genera Escherichia, Bacillus, andSalmonella as well as the genera Pseudomonas and Streptomyces. Intypical embodiments the host cell is of the genera Escherichia and canbe Escherichia coli (E. coli).

As shown in the Examples below, constructs of the invention provide foroptimal CAT deficient recombinant-protein expression under a variety ofconditions. These constructs are particularly efficient for expressionin prokaryotic hosts and in particular bacteria of the generaEscherichia. Note as well that various plant expression systems can alsobe used in the context of the present invention, typically usingAgrobacterium trameficies.

Endotoxin Free Fusion Protein Purification

Aspects of the present invention include use of endotoxin free,codon-optimized, CAT-deficient recombinant-protein for use invaccination of animals, and in particular for vaccination of farmanimals, which in some cases are United States bred dairy cows.Endotoxin free materials are particularly important for cattle bred andraised in the United States (see for example, Drackley, J K 2004.Physiological adaptations in transition dairy cows. Pp 74-87 in Proc.Minnesota Dairy Herd Health Conf., St Paul, Minn. University ofMinnesota, St. Paul). Also, because the methods contemplated hereininclude repeated vaccinations in animals, including humans, endotoxinfree compositions are of increased importance.

In one embodiment, the chimeric immunogenic recombinantprotein-comprising proteins of the invention are prepared bytransforming target cells with appropriate recombinantprotein-containing vehicles. As noted above, vehicles for use hereininclude known plasmid and vector systems suitable for expression inselected target cells.

In an aspect of the invention, chimeric immunogenic recombinantprotein-comprising proteins are expressed in target host cells. Chimericprotein expression is performed using target regulatory sequences. Insome aspects the chimeric polypeptides have been optimized (especiallywith regard to linker sequences disclosed herein) for expression in E.coli.

Chimeric protein can then be purified in accordance with known proteinpurification technologies, including, for example, lysozyme lysis,differential centrifugation of inclusion bodies, sieve chromatographyand the like. Refolding procedures can be conducted in guanidinechloride and urea at alkaline pH followed by dialysis andlyophilization.

In one embodiment, E. coli cells are transformed using acodon-optimized, CAT-deficient recombinant-protein containing plasmid,the plasmid having appropriate E. coli base regulatory sequences forexpression. In some cases, fermentation of approximately ten liters ofthese cells provides at least 500 grams and in some cases 600 grams oftotal biomass, yielding about 4-6 grams of total protein. It isestimated from silver and coomassie blue staining that up to half of theprotein can be chimeric protein (not shown).

In some embodiments herein, chimeric protein of the invention ispurified from transformed host cells in a substantially endotoxin freestate. Realization that endotoxin, and in particular multiple exposuresto endotoxin, in some animals, and in particular dairy cows, results insubstantially compromised animals (mastitis and endotoxin shock in dairycows bred and raised in the United States) was an unexpected andsurprising result that the present inventors obtained. This realizationresulted in an attempt to remove or lower the endotoxin dose amount ornumber of exposures in dairy cow vaccinations. Note that this endotoxinbased effect is much less realized in cows bred and raised in Russia andother countries as the dairy cattle are descendent from a differentstrain of cow (Holstein Association, 1 Holstein Place, Brattleboro, Vt.05302-0808). This finding in United States dairy cows is generallycontrary to the expectation that a vaccine should include some lowamount of endotoxin to help maximize an animals' immune response, as isthe case for dairy cows when vaccinated with somatostatin in some otherEuropean markets (see U.S. Pat. No. 6,316,004).

As such, some embodiments herein are directed at production ofsubstantially endotoxin free chimeric proteins for use in vaccines, andespecially for use in vaccines used in the cattle industry and used inthe cattle industry within the United States. In certain embodiments theendotoxin levels are at or below 1 EU/ml and in other embodiments theendotoxin levels are substantially eliminated, i.e., the chimericpolypeptides of the invention are substantially endotoxin free.

In one embodiment, recovered IP from lysed host cells is washed multipletimes using a wash solution devoid of endotoxin, i.e., endotoxin freewater or solution. The recovered IP pellet can optionally be washeduntil endotoxin levels are below approximately 1 EU/ml (endotoxin testscan be performed using one or more known assays, including commerciallyavailable test kits from MP Biochemicals, Charles River, etc.). In someembodiments the wash solution is endotoxin free and includes one or moreproteolytic protein inhibitor(s), e.g., phenylmethanesulphonylfluoride(PMSF), 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF), etc. In someembodiments the wash solution is phosphate buffered saline (PBS) havingan inhibitory effective amount of PMSF, AEBSF or a combination of bothPMSF and AEBSF.

In some aspects, substantially endotoxin free pellets can be treatedwith a protein unfolding solution at pH 12.5 containing urea andrefolded in a protein refolding solution containing a reduced molarityof urea with arginine, glycerol and/or sucrose. Purified chimericprotein concentration is modified to be between 1 and 3 mg/ml andtypically about 1.4 to 1.8 mg/ml. In some cases, substantially endotoxinfree chimeric protein is provided to vaccine formulations at about 1.5to 5 mg/2 ml dose and more typically from 2.0 to 3.5 mg/2 ml dose.

Other endotoxin removal procedures are envisioned to be within the scopeof the present invention and can include, for example, commerciallyavailable ion-exchange endotoxin removal columns, hydrophobic columns,etc (see for example Mustang E or G Columns (Millipore)).

Enhanced Immune Response Adjuvant

Embodiments of the invention provide new adjuvants for enhancedinduction of humoral immunity, or the aspect of immunity that ismediated by macromolecules found in extracellular fluids, such assecreted antibodies and antimicrobial peptides (as opposed tocell-mediated immunity, or the aspect of immunity relating tophagocytes, antigen-specific cytotoxic T-lymphocytes, and cytokineresponse). These adjuvants provide a significant improvement overconventional materials for the induction of a humoral response.Adjuvants herein can be used with numerous vaccines, but are shown inthe Examples in use with polypeptides of the invention for vaccinationin dairy cows, pigs or bull calves.

Importantly, all components of adjuvants herein are of non-animalorigin, thereby eliminating potential cross-contamination of vaccinatedanimals from potentially contaminated adjuvant components. For example,embodiments herein can utilize animal origin free Tween 80. This isparticularly important when the target animal is a dairy cow, due toconcerns over bovine spongiform encephalopathy (BSE) or other likebovine ailments. Note that these concerns are equally appropriate forhuman treatment where non-animal origin adjuvant provide significantsafety benefits. Additionally, adjuvant embodiments herein are free ofbenzene and other like carcinogenic compounds. These embodiments providea safety benefit not available in most conventional adjuvant compounds.For example, embodiments herein can utilize Carbopol® 974P or benzenefree polycyclic acid.

In one embodiment, the immunological adjuvant comprises an oil-in-wateremulsion in combination with selected antigens admixed within anemulsion premix.

Illustrative oil-in-water emulsions for use herein include combinationsof mineral oil, Tween 80, Span 85 and target polymers (benzene-freePolyacrylic acid). In some cases the target polymer is selected from thegroup consisting of Carbomer Homopolymer Type B. Typical oil-wateremulsions comprise from about 8-10% mineral oil (v/v), 0.003 to 0.004%Tween 80 (v/v), 0.007 to 0.008 Span 85 (v/v) and 0.04 to 0.06% polymer(w/v).

Illustrative emulsion premixes of the invention are composed of a highmolecular weight polymer, surfactant, and emulsifier in at approximate50% oil-aqueous base. High molecular weight polymers for use hereininclude acrylic acids crosslinked with allyl ethers of pentaerythritol.In some cases the high molecular weight polymers have a Brookfield RVTviscosity of between about 29,000 and 40,000, for example, Carbopol®974P (Noveon, Inc).

Methods for Treatment of Human Disease

The invention provides pharmaceutical grade vaccines containing chimericpolypeptides and adjuvants of the invention. Such vaccines can beadministered to patients having or at risk of contracting one or morediseases to cause an appropriate immunological response in a patient.Further, the methods of the invention allow vaccines to directlystimulate B cell response, improving response time and efficacy.

Vaccines of the invention are provided to patients having one or morediseases or deficiencies described herein. In one embodiment, vaccinesof the invention are provided 2 to 3 times to the patient in needthereof, with little or no anamnestic response observed. In otherembodiments, vaccines are provided 3 times or more, 4 or more, 5 ormore, or on a continual basis. In a preferred embodiment, vaccines arereadministered after a peak physiological effect or serum level isobserved. In other embodiments, vaccines are readministered after thephysiological effect is lessened or no longer observed, i.e. there hasbeen a return toward or to baseline. A typical vaccine antigen amountper administration is from 1 to 5 mg/ml chimeric polypeptide. Vaccinescan be administered by known techniques. In one embodiment the vaccineis administered via subcutaneous injection. In another embodiment thevaccine is administered by intradermal injection, intramuscularinjection or infusion.

Vaccine embodiments of the invention can further include dispersing orwetting agents, suspension agents, or other like materials. For example,embodiments can include sterile oils, synthetic mono- or diglycerides,fatty acids or oleic acids.

Vaccines are typically prepared as sterile, aqueous solutions. Thesesolutions are stable under conditions of manufacture and storage. Insome aspects, additional agents can be included in the vaccine toprevent microorganism action, for example, antibacterial or antifungalagents.

Vaccine solutions of the invention are prepared by incorporating thematerials (as described herein) in the required amounts (antigen,adjuvant, other ingredients) and can be followed by terminalsterilization, e.g., via UV light or ozone treatment. Alternatively,vaccine solutions of the invention can be prepared using individuallysterilized components prior to final assembly (in which case no terminalsterilization is required).

Treatment progress for patients receiving vaccine embodiments of theinvention can be monitored and additional administrations provided.Increase in deficient protein level and functional benefits (forexample, a decrease in disease symptoms) are all targets for monitoringtreatment effectiveness. In addition, levels of other markers can bemonitored to determine effectiveness of treatment on a patient.Anamnestic response in a patient may be determined by any of the abovemethods or by measuring the patient's antibody titers. Based on anindividual patients' progress, additional vaccine injections can beperformed using more or less antigen in accordance with the presentinvention. In some embodiments, vaccine injections may be performedrepeatedly if no anamnestic response is observed. In some embodiments,the injections are performed 3 times or more, 4 or more, or 5 or more.In other embodiments, the injections are performed continuously. In apreferred embodiment, vaccines are readministered after the peakphysiological effect or serum level is observed, or alternatively afterthe physiological effect has returned to baseline and is no longerobserved. In addition, alternative adjuvant combinations may be used tomodify a particular patients' response to vaccination, as determined bythe health care professional.

In some embodiments, the vaccinations are performed in an indefiniteseries, i.e. to treat a chronic disease or condition. Compositions ofthe invention are particularly useful for the long term treatment of adisease associated with a non-immunogenic protein, because the carriercomposition is capable of imparting immunogenicity to a protein whilenot generating a significant immune response to the carrier itself. Thisallows the compositions to be administered repeatedly and withoutneutralization. Examples of chronic diseases or conditions treated bythe methods described herein include growth disorders, immune disorders,cardiac disease, diabetes, stress disorders, or cancer. In someembodiments, the vaccine for the treatment of a chronic diseasecomprises a somatostatin-based target antigen.

Embodiments herein can be combined with other conventional therapies forthe target disease state or condition. For example, somatostatin-basedvaccinations of the invention can be combined with replacement insulinin the treatment of type 1 diabetes, or vaccinations can be combinedwith weight loss surgery or low calorie diets in a patient sufferingfrom severe obesity. In some embodiments, vaccinations of the inventionare combined with therapies that destroy or limit the effectiveness ofimmune cells or suppress immunological function. In a particularembodiment, vaccines of the invention are administered with therapiesfor autoimmune diseases (such as rheumatoid arthritis), allergies, Bcell lymphomas, or carcinomas.

In some embodiments, administration of B-epitope vaccines providedherein comprising a target antigen to which an immunogenic response isdesired may allow a patient with suppressed immune function to have anincreased immunological response to the protein, aiding in the treatmentof disease or preventing immunosuppression of a desired protein, such asan endogenous protein.

In one embodiment, the therapy is a stem cell therapy. In anotherembodiment, the therapy is a B cell depletion therapy that targetsmalignant B lineage cells. In other embodiments, the therapy is one thattreats a disease state by depleting B cells or reducing B cellactivation.

In some embodiments, a patient or agent of the patient identifies thebest recombinant protein and linker combination based on the specificdisease state and/or treatment goals. The recombinant protein isdesigned to allow a protein to bind to a specific linker and retain itsoverall conformation. In one embodiment, the recombinant protein isselected based upon a protein associated with the patient's diseasestate. In a particular embodiment, the recombinant protein is based upona protein that is upregulated or amplified in the patient as compared toa healthy individual. In another embodiment, the recombinant protein isbased upon a protein that is an agonist of or is upstream or downstreamof a protein that is upregulated or amplified in a disease state. In yetanother embodiment, the recombinant protein is based on a protein thatis an antagonist of a protein that is downregulated or inhibited in adisease state. The linker is selected based on the particular structuralcharacteristics of the carrier and recombinant protein. In someembodiments, the linker is selected for its B cell epitopes and abilityto activate B cells. In other embodiments, the linker is selected forits ability to connect to the recombinant protein without causing largechanges in the shape and conformity of the recombinant protein ascompared to the non-recombinant or naturally occurring protein that therecombinant protein is based on.

Method for Prime-Boost Vaccination

Embodiments of the invention provide novel methods of administering aprotein or antigen followed by administration of a vaccine comprising arecombinant version of the protein or antigen (i.e. a prime-boostvaccine). Proteins useful for this method include those containing Bepitopes, including but not limited to influenza neuraminidase, HIVgp120, dengue fever DEN-1 and 2, hepatitis B surface antigen,Staphylococcus aureus surface protein (MRSA), tetanus toxin, rabiesglycoprotein G, or malaria. In these embodiments, a patient is firstexposed to an immunogenic amount of a protein for which an immuneresponse is desired, for example to prevent the later development of adisease after exposure to an antigen or protein in the environment. Apatient is then administered the chimeric polypeptides of the invention,which are based on a recombinant version of the protein or antigenbelieved to cause the disease state. Administration of vaccinescomprising the recombinant protein generates antigenicity similar tothat of the original protein and allows the patient to quickly developimmune responses to subsequent presentations of the protein, preventingfuture disease states due to subsequent exposure.

In some embodiments, vaccines comprising the modular carrier-linker ofthe invention may be administered as a secondary or alternative boostervaccination where another vaccine has previously been administered. In apreferred embodiment, the vaccine of the invention is based on arecombinant version of a protein used in the original vaccination. Useas a secondary or alternative booster vaccination is particularly usefulwhere the immune system has generated immunological memory to thecarriers of the original or primary vaccine. Administering the vaccinesdescribed herein prevents subsequent neutralization of the carrier,allowing efficacious additional vaccinations with an antigen. In someembodiments, the vaccine is a administered as a secondary or alternativebooster vaccination where the primary vaccine is based on an antigencontaining B epitopes, including but not limited to including but notlimited to influenza neuraminidase, HIV gp120, dengue fever DEN-1 and 2,hepatitis B surface antigen, Staphylococcus aureus surface protein(MRSA), tetanus toxin, rabies glycoprotein G, or malaria.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 CAT-Defective Somatostatin Fusion Protein

Somatostatin (also known as somatostatin-14, growth hormone inhibitinghormone, or GHIH) is a peptide hormone produced in the hypothalamus aswell as certain portions of the digestive system. Somatostatin has twoactive forms that are produced by alternative cleavage of a polypeptide.Costoff A. Section 5, Chapter 4: Structure, Synthesis, and Secretion ofSomatostatin. Endocrinology: The Endocrine Pancreas. Medical College ofGeorgia, page 16, incorporated by reference in its entirety for allpurposes. Although it is contemplated that either somatostatin form canbe used in somatostatin-based antigen embodiments herein, and that theinvention includes additional antigens, somatostatin-14 will bedescribed in detail. Somatostatin-14 is a biologically activetetradecapeptide produced in the hypothalamus and gastrointestinal tract(stomach, intestine, and pancreas). The amino acid sequence of thetetradecapeptide is AGCKNFFWKTFTSC (SEQ ID NO: 1). The sequence ofsomatostatin-14 is highly conserved among vertebrates (Lin X W et al.Evolution of neuroendocrine peptide systems: gonadotropin-releasinghormone and somatostatin. Comp. Biochem. Physiol. C. Pharmacol. Toxicol.Endocrinol. 1998 119(3):375-88.) The tetradecapeptide is encoded by anucleic acid sequence: GCTGGCTGCAAGAATTTCTTCTGGAAGACTTTCACATCCTGT (SEQID NO: 15) (note that other nucleic acid sequences can be used to codeSEQ ID NO: 1, however, SEQ ID NO: 15 is provided for illustrativepurposes).

Somatostatin-14 is known to have a strong inhibitory effect on a largenumber of hormones involved in the growth and utilization of food inanimals, especially growth hormone and insulin. As previously describedin U.S. Pat. Nos. 6,316,004, 7,722,881 and U.S. Patent Publication No.2013/0149332 (incorporated herein by reference for all uses),somatostatin-14 and chimeric versions of somatostatin can be used inimmunization of animals for increase in daily weight and, whereappropriate, milk production. Unlike the current invention, theseimmunization procedures did not provide multiple administrations withoutanamnestic responses. Note that treatment of target animals withanti-somatostatin antibodies has proven to be overly costly andfunctionally non-dramatic, thereby eliminating direct antibody treatmentas non-practical. Muromtsev G. S., et al., 1990, Basics of agriculturalbiotechnology, Agropromizdat, Moscow, pp 102-106. One aspect of thepresent invention is based on the concept that the anti-somatostatinantibodies formed by compositions and methods described herein attenuatebut do not completely eliminate the mostly inhibitory actions ofsomatostatin in the target animal. This process produces a natural andproportional increase in growth and productivity in immunized targetanimals.

In particular, immunization of animals to somatostatin has beenrecognized as a means of neutralizing somatostatin in a target animaland thereby removing somatostatin's normal inhibitory effects on variousaspects of the animal's productivity, e.g. milk production in a dairycow (Reichlin S., ed., Somatostatin, Basic and Clinical Status. PlenumPress, New York 1987, pp 3-50, 121-36, 146-56, 169-82, 221-8, 267-74;Spencer, G. S., Review: Hormonal systems regulating growth. LivestockProduction Science 1985; 12:31-46. Importantly, these somatostatin-basedimmunization procedures avoid the direct use of anabolic hormones, e.g.growth hormone and the like, in the animal, and allow for small changesin the concentration of the endogenous anabolic factors and therebyprovide ecologically pure food products.

A number of studies have shown that animals immunized with somatostatinhave an average daily gain of 10-20%, an appetite reduced by 9% and an11% increase in the efficiency of food utilization. Animals immunizedwith somatostatin, and also their offspring, have correct proportions,and the distribution of the weight of the animals between the muscles,bones and fat is the same as in control animals (see Reichlin, 1987).Therefore, somatostatin immunization provides a useful and safe way ofenhancing a target animal's productivity. This is particularly the casewhen compared with use of recombinant growth hormone, which use hasraised concerns over hormone in milk or meat from treated animals or forthe safety of the animals themselves or for build-up of the hormone inthe ecosystem, particularly the ground water supply.

Somatostatin is known to have a relatively short half-life in the blood.In order to enhance the immunologic effects of somatostatin,immunization protocols have been developed to enhance the protein'shalf-life by conjugating somatostatin to target carrier proteins. Theseconjugated somatostatin proteins are designed to have increasedhalf-life and increased antigenicity in the blood and therefore provideenhanced benefits, especially in light of the cost of preparingsomatostatin. For example, chimeric somatostatin proteins are disclosedin U.S. Pat. No. 6,316,004, corresponding European Patent EP0645454, andU.S. Pat. No. 7,722,881, each of which is incorporated herein byreference, where various conjugated somatostatin-containing proteins areshown to have increased antigenicity and function with regard toproductivity of farm animals as compared to other conventionalimmunization or anabolic hormone-based procedures. U.S. PatentPublication No. 2013/0149332 discloses similar compositions and showstheir beneficial use in other diseases, including human obesity.

These methods provide an unexpected improvement over other methods ofadministering vaccines.

The present example illustrates the production of a CAT-defectivesomatostatin fusion protein in accordance with embodiments of thepresent invention. Site-directed mutagenesis was performed on plasmidpET30b-Cat-Som to replace His192 and His193 with glycine residues (aftermodification: Gly192 and Gly193). Inactivation of the His193 (andHis192) residues eliminates the capacity of the CAT enzyme to acceptprotons, thereby providing complete inactivation of the CAT.

The linker in the same pET30b-Cat-Som (having the His replacement(s))was codon-optimized for expression by E. coli in the absence ofco-expressed tRNA molecules.

The modified CAT-defective somatostatin nucleic acid construct is shownas SEQ ID NO: 12. The CAT-defective somatostatin fusion protein sequenceis disclosed as SEQ ID NO: 13, being compared to an unmodifiedCAT-somatostatin fusion protein (SEQ ID NO: 14).

Example 2 CAT-Defective Somatostatin Fusion Protein can be Expressed atHigh Levels

The codon-optimized CAT-defective somatostatin construct as described inExample 1 was used to express the fusion protein in BL21(DE3) cells.Transformed cells were grown in LB and induced with 0.4 mM IPTG forapproximately three hours. One milliliter of cells from a density of OD0.7 culture were pelleted, and heated at 70° C. for ten minutes in 100μl SDS sample buffer. A sample of 40 μl of cell extract was loaded perlane for SDS PAGE.

28 KD band corresponding to the predicted size of a codon-optimized,CAT-defective somatostatin fusion protein was visible in lanes 1(LB+IPTG, reduced) and 3 (LB+IPTG) after induction with IPTG. Noexpression is seen in control lanes 2 (LB, reduced) and 4 (LB). Asexpected, there was no difference in fusion protein size when run understandard or reducing conditions.

Example 3 Endotoxin Free, Codon-Optimized CAT-Deficient SomatostatinContaining Vaccine

An illustrative vaccine comprising the fusion protein of the previousexamples in accordance with the present invention:

Reagent Solution:

-   -   1. Carbopol Base        -   a. Dissolve 0.5 grams of Carbopol 974P in water or saline        -   b. Mix and boil to dissolve. Followed by autoclaving.        -   c. Store at 4° C.    -   2. Squalene Base        -   a. Mix 58.1 ml of squalene, 4.6 ml of non-animal origin            Tween 80 and 5.2 ml of Span 85.        -   b. Mixture was filtered through a 0.2μ filter.        -   c. Store at 4° C.    -   3. Tragacanthin solution        -   a. Extract tragacanth gum with methanol.        -   b. Collect methanol insoluble fraction.        -   c. Dry at room temperature.        -   d. Store at room temperature in a desiccated state,        -   e. Add 1 gram of dried Tragacanthin in water or saline.        -   f. Mix and boil to dissolve, followed by autoclaving.        -   g. Store at 4° C.            Vaccine Preparation    -   1. Vaccine antigens are prepared in saline or PBS at 5 mg/ml or        lower.    -   2. Add 6.79 ml of squalene base to mixing bottle.    -   3. Add 10 ml of Carbopol base to Squalene base. (CS)    -   4. Mix well.    -   5. Add 10 ml of Tragacanthin solution to CS solution.    -   6. Mix well.    -   7. Vaccine antigens, undiluted or diluted to use in saline or        PBS, are added to a final volume of 82 ml.    -   8. 1 ml of a 1% Thimerosal solution is added and mixed well.    -   9. Store vaccine at 4° C. until use.

-   Alternative illustrative vaccine in accordance with the present    invention:

Reagent Solutions:

-   -   1. Carbopol Base:        -   a. Dissolve 0.5 grams of Carbopol 974P in water or saline;        -   a. Mix and boil to dissolve; and autoclave        -   b. Store at 4° C.    -   2. Squalene Base:        -   a. Mix 58.1 ml of squalene, 4.6 ml of non-animal origin            Tween 80 and 5.2 ml of Span 85; and filter through 0.2μ            filter        -   b. Store at 4° C.    -   3. Arabinogalactan solution:        -   a. Add 1-10 grams of arabinogalactan into PBS or saline;        -   b. Mix and boil to dissolve; and autoclave        -   c. Store at 4° C.

Vaccine Preparation:

-   -   1. Vaccine antigens are prepared in saline or PBS at 5 mg/ml or        lower;    -   2. Add 6.79 ml of squalene base to mixing bottle;    -   3. Add 10 ml of Carbopol base to the Squalene base;    -   4. Mix thoroughly and add 10 ml of arabinogalactan solution;    -   5. Antigens of the invention, undiluted or diluted, to use in        saline or PBS, are added to a final volume of 82 ml.    -   6. 1 ml of a 1% thimerosal solution is added and the vaccine        mixed; and    -   7. The vaccine is stored at 4° C. until use.

Example 4 Treatment of Cardio Vascular Disease Using Vaccine of Example3

The present Example uses rats with left ventricle dysfunction asprepared in the protocol published in Genentech, 1995. Two groups ofrats are segregated (each member of each group having a ligated leftcoronary artery), a first treatment group receives the vaccinations ofthe invention and a second control group (no vaccination, but otherwisetreated the same). Each member of the treatment group receives avaccination and then 21 days later a second vaccination and a thirdvaccination at 42 days, administered intramuscularly (1 ml/dose). SerumIGF-1 levels and anti-somatostatin antibodies are measured at day 0, day21, day 42 and day 63. At day 63, hemodynamic parameters are alsomeasured in both groups as well as a determination of infarct size andcardiac index.

It is anticipated that the rat group receiving the three vaccinations,as described in Example 3, would have substantially improved cardiacfunction (decrease infarct size and improved cardiac index) as comparedto the control group. It is also anticipated that rat groups in furtherstudies, in which the vaccinations are repeated at regular intervalsover a substantial or indefinite period of time, would continue to havesubstantially improved cardiac function as compared to the controlgroup. This makes vaccines of the invention particularly useful in thelong-term treatment of cardiovascular disease.

Example 5 Treatment of Growth Deficiency Using Vaccine of Example 3

Three week old Cox (CD) rats will be vaccinated monthly for 3 monthsusing a 1 ml dose. Each vaccination will occur intramuscularly orsubcutaneously. Control rats will receive saline injections, using thesame mode of administration and the same volume of material foradministration. All rats will be weighed to determine growth on a weeklybasis and bled at 0, 4, 8, 12 and 16 weeks. Serum will be collected at asimilar schedule and analyzed for IGF-1, urea and anti-somatostatinantibody levels.

It is expected that CD rats receiving vaccinations as described inExample 3 will have substantially improved growth as compared to controlCD rats. Treated rats should show serum results that confirmvaccinations effects on treated rats. It is also anticipated that ratgroups in further studies, in which the vaccinations are repeated atregular intervals over a substantial or indefinite period of time, wouldcontinue to have substantially improved cardiac function as compared tothe control group. This makes vaccines of the invention particularlyuseful in the long-term treatment of growth deficiency.

Example 6 Treatment of Obesity in Mice

Mouse obesity studies were performed using mice from JacksonLaboratories, Bar Harbor, Me. A number of inbred mice from line C57BL/6Jwere obtained from Jackson Laboratories, the mice were: male, showedinduced severe obesity, had polygenic genetics, and exhibited matureonset obesity. In previous testing, Jackson Laboratories had determinedthat this particular strain of mice, when fed on a high fat diet,develops metabolic syndrome phenotypes very similar in nature to thosereported in the human population. For example, C57BL/6J mice fed a highfat diet will show visceral adiposity, insulin resistance,hyperinsulinemia, hyperleptinemia, leptin resistance and hypertension.

Studies were conducted to test the effectiveness of vaccine embodimentsherein for treating obesity, i.e., including limiting weight gain insome mice to causing weight loss in C57BL/6J mice. Six week old micewere fed a 60% kcal % fat diet for 6 weeks. Twelve week old mice werethen broken into one of four groups: group 1 included mice treated withJH14 containing vaccine; group 2 included mice treated with JH17containing vaccine, group 3 included mice treated with JH18 containingvaccine, and group 4 included control mice that were treated with PBSrather than any type of anti-somatostatin type antigen. Mice in eachgroup were vaccinated using a 0.5 ml of the specified vaccine or PBS viaan IP route. After twenty two days the mice were treated again with asecond IP dose using 0.1 ml of vaccine.

Throughout the course of the study (6 weeks) each mouse was weighed twotimes per week and food intake monitored, i.e., to ensure that weightchanges were not due to loss or increase in food intake. A terminalbleed was performed on each mouse at the conclusion of the study andIGF-1 levels determined (IGF-1 plasma levels were determined usingDiagnostic Systems Laboratories Inc. Active Mouse/Rat IGF-1 ELISA(DSL-10-29200).

As shown in Table 3, mice treated with JH14, 17 and 18 all showed ahighly significant difference (p<0.0001) by parametric or non-parametricstatistical analyses) in percent Final Body Weight vs. Baseline Weight.Significant weight loss was observed in each vaccinated group within thefirst 7 days while the control group showed slight weight gain over thesame time period. A small weight loss was also observed after the seconddose of vaccine (⅕ dose provided on day 1) was administered to the JH14,JH17 and JH18 groups at day 22.

Data from the mouse obesity study provided the following conclusions:(1) although there is not a statistically significant difference betweenJH18 and the controls, in terms of IGF-1 ng/ml, there is a highlysignificant difference between these groups (P<0.0001) by parametric ornon-parametric statistical analysis in percent Final Body Weight versusBaseline Weight; (2) In percent Final Body Weight versus BaselineWeight, JH17 versus the controls produced a statistically significantdifference by both statistical tests; (3) JH18 (which had a mean IGF-1level of 135.8 ng/ml more than JH17), demonstrated a statisticallysignificant difference versus JH17 in percent baseline weight (only bythe non-parametric test); (4) chimeric-somatostatin antigen of theinvention in both JH17 and JH18 adjuvants induced a statisticallysignificant difference in percent Final Body Weight versus BaselineWeight; (5) JH18 was statistically significant when compared with JH17by non-parametric analysis in terms of percent Final Body Weigh versusBaseline Weight; (6) IGF-1 levels can be correlated with a greaterweight loss at the end of the study versus both controls and JH17vaccinates (see Table 4); (7) since all vaccinates had the same doseamounts of the chimeric-somatostatin antigen of the invention, anadjuvant affect was observed within the study; (8) inbred C57BL/6J malemice fed 60% kcal fat diet demonstrated a significant weight loss withinthe first week post IP vaccination; and (9) the weight loss shown hereinpersisted even while the mice ate a 60 kcal % fat diet for the durationof the study.

TABLE 3 Final Body Weight versus Baseline Weight Mann Unpaired %Standard Whitney t-test Group # Baseline Deviation (two tailed)(two-tailed) Controls 10 115.5 6.3 Not Done Not Done JH17 10 107.1 4.7 P= 0.0021 P = 0.033  JH18 10 104 3.0 P < 0.0001 P < 0.0001 JH17 vs. JH18— — — P = 0.0355 P = 0.1016

TABLE 4 IGF-1 Statistical Analysis Mean IGF-1 Standard Mann WhitneyGroup # (ng/ml) Deviation (1 tailed) Controls 10 365.6 88.7 Not DoneJH17 10 304.2 99.2 P = 0.0827 JH18 10 440.4 103.7 P = 0.105 

Example 7 Endotoxin Free Chimeric Peptides/Adjuvants Provide IncreasedMilk Production

A random pool of dairy cows (Holstein Crosses—US bred and raised) wasidentified, each was 31 to 65 days post-calving (3^(rd) through 5^(th)lactation). Each cow was examined and determined to be in optimal healthby a veterinarian.

The average cow weight in the study was from about 1,000 to 1,200 lbs.Six lactating cows were treated with 1.96 mg/chimeric protein/2 ml dosein JH14. Alternatively, 9 lactating cows were provided with aconventional rB ST treatment. Treatments and milk production study wasconducted at a large scale, intense milk production dairy.

Vaccinations were conducted at day 0. Anti-SST serum antibodies andIGF-1 serum levels tested at 4 weeks. Milk production and identificationof general health of animals were conducted on a regular schedule.

Six cows that were vaccinated using inventive compositions describedherein had a normal appearance, with no endotoxin reaction or foodwithdrawal. All six cows had a positive serologic response to SST with amean titer of 1:14. Milk production of the six cows was obtained withonly one vaccination, showing a mean yield increase of 23.7%.

Nine cows treated using conventional rBST injections at 0 and 14 dayswith an overall mean increase in milk productivity of 2%.

The data in this Example shows the drastic improvement in effectivenessfor using the endotoxin free constructs in combination with inventiveadjuvants in dairy cows. These results are dramatically improved towardthe animal's health and productiveness as compared to cows rejected twotimes with rB ST.

Example 8 Multiple Vaccinations with Chimeric Peptides/AdjuvantsIncrease Milk Production

A pool of 92 clinically healthy dairy cows (Primiparous and MultiparousGirilander and Girilander/Holstein crosses) was identified, each was 90to 120 days post-calving. The cows were divided into four treatmentgroups, with one receiving a single vaccine dose, one receiving a doubledose, one receiving a quadruple dose, and a saline control group.Treatments and milk production study was conducted at a large scale,intense milk production dairy.

Cows were vaccinated intramuscularly in the neck region on days 0, 21and 42 of the study, with the injection site alternating from right toleft to right. The cows were milked 3 times a day according to thefarm's practices, and milk yields were recorded for each cow. Milk wasanalyzed for milk fat, lactose, protein, urea and Somatic Cell Count(SCC). Cows were observed daily for injection site reactions, generalhealth, mastitis and foot problems, and body scores were obtained at 21day intervals. The study concluded 21 days post 3^(rd) vaccination, or63 days from its start.

The treated cows showed increased milk yields compared topre-vaccination yields, with increases beginning by day 4post-vaccination and persisting through day 21, with peak yields 8-14days post vaccination. As shown in FIGS. 3 and 4, this trend wasrepeated upon each of the three vaccinations. As compared with controlcows, vaccinated cows demonstrated an increase of 5-20% in milkproduction in the post vaccination periods, with a mean increase in milkyield of 13.6%. Using an LSmeans model, there was a highly statisticaldifference in milk production between treatment weeks.

No significant differences were observed among the treatment groups withrespect to site reactions, body scores, general health, mastitis or footproblems. Vaccination, even at a quadruple dose, was well tolerated bythe cows and produced no untoward effects as compared to the controls.

The data in this Example shows the surprising finding that multiplerepeated injections of the compounds of the invention are highlyefficacious for rapidly increasing milk production, and that theincreased production may be sustained using multiple injections over alengthy period. It would be expected that multiple or repeat injectionsat regular intervals over a substantial or indefinite period of timewould continue to cause improved milk production. This makes vaccines ofthe invention particularly useful in the long-term improvement of milkproduction.

Example 9 Endotoxin Free Chimeric Peptides/Adjuvants Provide IncreasedMeat Production in Piglets of Treated Sows

A random pool of sows will be identified, each being at least 35-36 daysprior to farrowing. Each sow will be examined and determined to be inoptimal health by a veterinarian. Pregnant sows will be immunized twotimes using vaccines of the invention (see Example 3), once at 35-36days prior to delivery, and once at 8 days prior to delivery. A controlgroup of pregnant sows will be maintained for comparison purposes (novaccinations or vaccination with sterile saline).

Delivered piglets from the vaccinated group will have greatersurvivability and be of a greater average size. It is the increase inpiglet size that enhances the percent survivability, as larger pigletsare less likely to be pushed away from the sow's teat. Treated andcontrol piglets will be weighed at day 21, day 30 and day 75.Vaccinations of the present invention will increase the piglet dailyweight by an average of 35% over the course of the 75 day period.

Importantly, piglet survivability and weight are increased through useof the vaccines of the present invention in the absence of recombinantgrowth hormone. This is a significant improvement over recombinanthormone therapy.

Example 10 Endotoxin Free Chimeric Peptides/Adjuvants Provide IncreasedMeat Production in Treated Pigs

36 barrows of pigs were purchased from a commercial source as feederpigs, each weighing approximately 22 kg. After a one week acclimationperiod, the pigs were fasted overnight and then weighed to establishweight blocks. Pigs from each of the weight blocks were allotted atrandom to three treatment groups as follows: saline control (controlgroup), JH14 adjuvant containing 0.1 mg/ml of recombinant Human SerumAlbumin (rHSA) adjuvant (adjuvant group), and JH14 adjuvant containing0.5 mg/ml of SST-CAT chimeric protein (vaccine group). The pigs werehoused in individual pens (n=12) and provided feed and water ad libitum.A common corn-soybean meal-based diet was fed to both treatment groupsand formulated to ensure that inadequate nutrient supply does not impairanimal response to treatment.

Initial blood samples for serum recovery were obtained by jugularvenipuncture. All vaccinations were with 1 ml administeredintramuscularly. Subsequent vaccinations were administered at 4 and 8weeks, and blood samples were collected bi-weekly for serum recovery,serum hormone and metabolite measurement, and to measure antibodytiters. Serum metabolites (NEFA, triglycerides, glucose and ureanitrogen) were measured in an auto-analyzer in a clinical pathologylaboratory, while porcine insulin, IGF-1 and somatostatin were measuredby commercially available ELISA kits. Serological responses weremonitored by ELISA utilizing rCAT, sSST and rHSA as coating antigens.Body weight and feed intake data were also collected bi-weekly todetermine average daily gain. At the conclusion 12 weeks after initialvaccination, final body weight and feed intake measures were taken afteran overnight fast, and final blood samples were obtained. Thereafter,pigs were allowed to consume feed ad libitum until three hours prior toeuthanasia on the following day, followed by processing in a certifiedprocessing facility.

TABLE 5 Differential weight gain (Δ gain) by VAX group pigs compared tocontrol. Weeks Post 3^(rd) Mean Δ Time Period Vaccination gain (kg) Week8 0 0 Week 9 1 2.3 Week 10 2 2.34 Week 11 3 2.97 Week 12 4 3.3

As compared to controls, vaccination group pigs demonstrated an increasein weight gain post 3^(rd) vaccination. Overall, three was a significantincrease in body weight over the 12 week study, as shown in Table 5 andFIG. 5A. The increase was particularly large in the final four weeks(FIG. 5B). FIG. 5C shows the difference in mean body weight betweenvaccinated and control pigs.

The cumulative weight differences for the entire 12 week study periodwere 3.4 kg, as shown in FIG. 5C.

As shown FIG. 6, ELISA responses to the indicator control antigen (rHSA)were similar to the responses to rCAT, indicating normal immune functionin the vaccination group. However, responses to CAT did not demonstrateenhancement until after the 3^(rd) vaccination (week 8), and unlike rHSAdid not demonstrate a titer drop (plateau effect) post 10 weeks. Of the12 pigs in the vaccination group, positive serological responses wereobserved in 100% of the pigs from week 8 through week 12. The responsesto CAT were similar to Human Serum Albumin (HSA) vaccines, but thesomatostatin responses were somewhat different.

In the same period, serological responses to rSST are shown in FIGS. 7and 8. Surprisingly, 100% seroconversion was seen from week 4 to week12, as shown in FIG. 7. Significant increases in anti-SST IgG weredetected at weeks 8, 10 and 12, as shown in FIG. 8.

The study results did not indicate any deleterious side effects due tovaccination, and injection site observation at processing did notindicate any macroscopic pathology or meat quality issues. Serummetabolite and hormone levels, determined at 12 weeks and shown in Table7 indicated no statistically significant changes in the vaccinated groupas compared to control pigs.

TABLE 6 Summary of weight gain in piglets CONTROLS VACCINATES (n = 10)(n = 10) Final Weight (kg) 127.5 130.7 Gain from week 0 (kg) 97.6 101.03Body Mass index 58.4 60.4

TABLE 7 Pig measurements at 12 weeks of the study (4 weeks post 3^(rd)Vaccination) Hormone/Metabolite Controls Vaccinates Somatostatin (pg/ml)24.9 27.3 Serum Triglycerides (mM) 1.8 2.4 Serum IGF-1 (ng/ml) 23.6 22.2NEFA (mM) 0.39 0.40 Glucose (mg/dL) 103.39 113.35 Serum Urea (mg/dL)60.5 54.3 Insulin (ng/ml) 0.247 0.384

The data in this Example shows that multiple repeated injections of thecompounds of the invention are highly efficacious for improving meatproduction, and that the increased production may be sustained usingmultiple injections over a lengthy period. The lack of an observedboosting effect due to endogenous somatostatin results in an improvedability to repeatedly vaccinate the animals, resulting in improvedproduct yields.

Example 11 Endotoxin Free Chimeric Peptide/Adjuvants Provide IncreasedMeat Production in Treated Bull Calves

A random pool of bull calves, one to three months of age, will beidentified, and injected with compositions of the invention. Weightincrease over a period of approximately ten months will be monitored andcompared to a control group, the control group being treated the same inevery sense as the injected group except for the vaccine injections ofthe invention. Each bull calve will be examined and determined to be inoptimal health by a veterinarian over the course of the treatments.

Injections herein for the vaccinated group are performed at zero weeks,4 weeks and 8 (three total vaccinations). Vaccinations will be providedsubcutaneously or intramuscularly to the neck using 18-21 gauge ccneedle. Booster injections were also provided (4 boosts, three boosts orno boosts). Vaccination injections included 2 mg/2 ml of the chimericpolypeptide. The chimeric polypeptide was prepared as described hereinhaving both histidine residues in CAT replaced with glycine amino acidsof the invention and an optimized linker as described by SEQ ID NO: 4.

Vaccinated bull calves and control calves are each weighed to take aninitial weight. It is expected that vaccinated animals herein will showa 15 to 40% weight increase over control animals. This increase inaverage weight for treated bull calves shows a significant advantageover no treatment.

Importantly, harvested meat from treated bull calves does not containrecombinant growth hormone.

Example 12 B Cell Epitope Prediction for Polypeptide Conjugates

The BepiPred 1.0 method was used as described by Larsen, Ole Lund andMorten Nielsen, “Improved method for predicting linear B-cell epitopes”,Immunome Research 2:2, 2006, which is incorporated herein by reference,and as found in http://www.cbs.dtu.dk/services/BepiPred/, to predictB-cell epitopes in a polypeptide conjugate and component target antigen,carrier polypeptide, and linker polypeptides. Model predictions wereinitially based on amino acid sequences disclosed in ChloramphenicolAcetyl Transferase (CAT)-Defective Somatostatin Fusion Protein disclosedin U.S. Pat. Nos. 7,722,881 and 8,425,914, each of which is hereinincorporated herein by reference. The following sequences were used inthe calculations.

Model target antigen: somatostatin 14: SST: (SEQ ID NO: 1)AGCKNFFWKTFTSC (14 Aas) Model linker polypeptide: (SEQ ID NO: 10)welhrsgprprprprpefm (19 Aas)Model carrier polypeptide: inactivated CAT enzyme(corresponding to his→gly at both 192 and 193): (SEQ ID NO: 3)Mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqv

avcdgfh vgrmlnelqq (210 Aas) Model linker-SST: (SEQ ID NO: 30)welhrsgprprprprpefmAGCKNFFWKTFTSC (33 Aas)Model polypeptide conjugate: carrier-linker-SST: SEQ ID NO: 13(SEQ ID NO: 13) mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvggavcdgfhvgrmlnelqqwelhrsgprprprprpefmagcknffwktftsc (243 Aas)

Employing these sequences, predicted B-cell epitopes were calculatedusing the Bepipred 1.0 method. Results are shown in Table 8.

TABLE 8 B-cell epitope scores for Polypeptide Conjugate. # # > 0.2Linear B Protein AA Threshold Epitopes SST (target antigen) 14 0   0%(SEQ ID NO: 1) Linker 19 16 84.2% (SEQ ID NO: 10) CAT inactivated 210 2813.3% (carrier) (SEQ ID NO: 3) Linker-SST 33 16 48.5% (SEQ ID NO: 30)CAT-linker-SST 243 45 18.5% (SEQ ID NO: 13)

A graph of the predicted B-cell epitope characteristics along thesequence of the polypeptide conjugate of SEQ ID NO: 13 is shown in FIG.9. Remarkably, the linker having high % predicted linear B-cell epitopesimparts enhanced B-cell epitope characteristics to the polypeptideconjugate which results in improved immunogenicity of the targetantigen, and improved immunological response/effect in the animalfollowing administration, for example, as shown in improved rapid milkproduction, as shown in FIG. 4.

While the invention has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the invention and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims.

What is claimed is:
 1. A method for rapidly increasing milk productionin an animal, the method comprising: administering an immunogenic amountof a vaccine to an animal, the vaccine comprising a polypeptideconjugate comprising a target antigen that is somatostatin-14 comprisingthe amino acid sequence of SEQ ID NO: 1, attached to an inactivatedchloramphenicol acetyl transferase (CAT) enzyme by a linker polypeptide,wherein milk production is significantly increased within 4 days of theadministration of the vaccine, relative to that of a control animal thatdoes not receive administration of said vaccine.
 2. The method accordingto claim 1, wherein the animal is a dairy cow, beef cow, goat, or sow.3. The method according to claim 1, wherein peak milk production isachieved within 8-14 days of the administration of the vaccine.
 4. Themethod according to claim 1, wherein the increase in milk productionpersists for at least 21 days.
 5. The method according to claim 1,further comprising administering an additional immunogenic amount of thevaccine to the animal, wherein the additional administration isperformed after peak milk production has been observed.
 6. The method ofclaim 5, wherein the additional administration is performed after areturn to baseline milk production has been observed.
 7. The method ofclaim 6, wherein the additional administration is performed at intervalsof approximately 21 days.
 8. The method of claim 5, wherein subsequentadministration of the vaccine does not produce an anamnestic response inthe animal.
 9. The method according to claim 1, wherein the inactivatedCAT enzyme has one or more of 192 or 193 wild type histidine residuesindependently replaced with an amino acid selected from alanine,glycine, or other like amino acid.
 10. The method according to claim 1,Wherein the inactivated CAT enzyme is truncated at the C-terminus by 10amino acids relative to the wild type polypeptide.
 11. The methodaccording to claim 1, the inactivated CAT enzyme comprising the aminoacid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 8,26, 27, 28 and
 29. 12. The method according to claim 1, the linkerpolypeptide comprising the amino acid sequence repeat of (Arg-Pro)_(n)or (Lys-Pro)_(n), where n=2 to
 6. 13. The method according to claim 1,the linker polypeptide comprising the amino acid sequence selected fromthe group consisting of SEQ ID NO: 10, 11, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 53, 54, 55, 56 and
 57. 14. The method according to claim 1,the polypeptide conjugate comprising the amino acid sequence of SEQ IDNO:
 13. 15. The method according to claim 1, wherein the vaccine furthercomprises an adjuvant.
 16. The method according to claim 15, wherein theadjuvant is an oil-in-water adjuvant.
 17. The method according to claim15, wherein the adjuvant comprises a carbopol and a squalene.
 18. Themethod according to claim 17, wherein the adjuvant further comprises anarabinogalactan, or a tragacanth.
 19. The method according to claim 1,wherein the immunogenic amount comprises a 1-2 mL dose of from 0.5-5mg/mL of the polypeptide conjugate.